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1.
Eur Rev Med Pharmacol Sci ; 26(16): 5689-5697, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36066141

RESUMO

OBJECTIVE: This study aims to summarize the role of PIWIs/piRNAs in cell apoptosis through multiple signaling pathways. The PIWI-interacting RNAs (piRNAs) are among the small non-coding RNAs (sncRNAs) and are mainly expressed in germline cells. PIWI protein is the key to the biogenesis of piRNA. With the deepening of research in recent years, the PIWIs/piRNAs are expressed in a tissue-specific way in somatic cells outside the germline. In addition, researchers have found that the PIWIs/piRNAs play a regulatory role in cell apoptosis, proliferation, and necrosis by regulating key signaling pathways, such as PI3K/Akt signaling pathway, STAT signaling pathway, TGF-ß signaling pathway, and Fas signaling pathway at the transcriptional or post-transcriptional level. However, the PIWIs/piRNAs' role in cell apoptosis and its underlying mechanisms are still not fully understood. This study reviews the regulatory functions of PIWIs/piRNAs in apoptosis from the perspective of the signal pathway. MATERIALS AND METHODS: This study is a narrative review. PubMed and MEDLINE were used as the primary sources to search the following keywords: PIWI/piRNAs, signal pathway, pro-apoptotic, anti-apoptotic, and signaling pathway. RESULTS: PIWIs/piRNAs modulated pro-apoptotic or anti-apoptotic effects in a variety of cells: PIWIs/piRNAs through PI3K/Akt signaling pathway, STAT signaling pathway, TGF-ß signaling pathway, and Fas signaling pathway for pro-apoptotic or anti-apoptotic effects in cells. CONCLUSIONS: Apoptosis is a basic biological phenomenon of cell death, and it also has a great significance and complex molecular biological mechanisms. PIWI/piRNAs are closely related to various types of diseases and play a pro-apoptotic or anti-apoptotic role through the following pathways: PI3K/Akt signaling, STAT signaling, TGF-ß signaling, and Fas signaling pathways.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Apoptose , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta
2.
Cardiovasc Ther ; 2022: 9729018, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36082193

RESUMO

Ischemia/reperfusion (I/R) injury is accompanied by an increase of matrix metalloproteinase 2 (MMP-2) activity, which degrades heart contractile proteins. The aim of the study was to investigate the effect of MMP-2 small interfering RNA (MMP-2 siRNA) administration on I/R heart. Isolated rat hearts perfused by the Langendorff method were subjected to I/R in the presence or absence of MMP-2 siRNA. The hemodynamic parameters of heart function were monitored. Lactate dehydrogenase (LDH) activity was measured in coronary effluents. Activity and concentration of MMPs in the hearts were measured. Concentration of troponin I (TnI) in coronary effluents was examined as a target for MMP-2 degradation. Recovery of heart mechanical function was reduced after I/R; however, administration of MMP-2 siRNA resulted in restoration of proper mechanical function (p < 0.001). LDH activity was decreased after the use of MMP-2 siRNA (p = 0.02), providing evidence for reduced cardiac damage. Both MMP-2 and MMP-9 syntheses as well as their activity were inhibited in the I/R hearts after siRNA administration (p < 0.05). MMP-2 siRNA administration inhibited TnI release into the coronary effluents (p < 0.001). The use of MMP-2 siRNA contributed to the improvement of heart mechanical function and reduction of contractile proteins degradation during I/R; therefore, MMP-2 siRNA may be considered a cardioprotective agent.


Assuntos
Metaloproteinase 2 da Matriz , Traumatismo por Reperfusão Miocárdica , Animais , Coração , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Troponina I/genética
3.
Contrast Media Mol Imaging ; 2022: 7511345, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072628

RESUMO

Background: Recently, inflammation has become a major threat to human health. Studies have confirmed that some Chinese traditional medicine ingredients may effectively interfere with the expression of inflammatory mediators through epigenetic modification, showing a great potential of the application. Objective: To investigate the role of the PPAR/DNMT3A pathway in the reversal of galangin-mediated inflammatory lung injury, promote the development of new anti-inflammatory drugs, reduce the side effects of chemical synthetic drugs on the body, and prove the effectiveness and safety of galangin in inhibiting inflammatory response and injury. Methods: 120 rats were randomly divided into 6 groups: (Group 1) LPS group; (Group 2) LPS + galangin group; (Group 3) LPS + galangin + GW9662 group; (Group 4) LPS + galangin + DNMT3A siRNA group; (Group 5) LPS + galangin + siRNA negative group; (Group 6) control group. The model of inflammatory lung injury was established by intrathecal instillation of LPS in the first five groups and NS in the control group. SD survival rate was recorded every 24 hours after modeling, lasting for 168 hours. The lung tissues were taken 168 hours after the establishment of the model. The pathological morphology of lung tissue was observed after the staining under the light microscope, and the lung dry/wet weight ratio was calculated after drying. After NS was perfused into lung tissue, the lavage fluid was collected and the levels of IL-6 and TNF-a were measured by ELISA. The contents of PPAR, DNMT3A, phosphorylated p65, and ERK in monocytes were detected by the WB method, and the binding contents of p65 and AP-1 in the promoter regions of IL-6 and TNF-a genes were detected by the Chip-qPCR method. Results: Intraperitoneal injection of galangin could inhibit the synthesis of alveolar inflammatory factors (TFs) in the SD model of lung injury induced by LPS, reduce the degree of pathological injury of lung tissue, and improve the survival rate of the SD model. GW9662 can completely reverse the protective effect, while DNMT3A interference can only partially block its protective effect. In addition, galangin could significantly inhibit the LPS-induced expression of p65 and AP-1 in alveolar monocytes and their binding content in the promoter region of inflammatory genes by activating PPAR/DNMT3A pathway. GW9662 could completely reverse the inhibitory effect of galangin. DNMT3A interference could restore the binding content of transcription factors at the promoter of the inflammatory gene but had no significant effect on its synthesis. Conclusion: Galangin can interfere with the binding of transcription factors to inflammatory gene promoters through the methylation modification induced by PPAR/DNMT3A pathway, so as to inhibit the synthesis of inflammatory molecules and reverse inflammatory lung injury.


Assuntos
Lesão Pulmonar Aguda , Flavonoides , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Flavonoides/efeitos adversos , Interleucina-6/metabolismo , Lipopolissacarídeos , Metilação , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Transcrição AP-1/metabolismo
4.
Int J Mol Sci ; 23(17)2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36077117

RESUMO

Interleukin-23 (IL-23) plays a pivotal role in rheumatoid arthritis (RA). IL-23 and microRNA-223 (miR-223) are both up-regulated and mediate osteoclastogenesis in mice with collagen-induced arthritis (CIA). The aim of this study was to examine the association between IL-23 and miR-223 in contributing to osteoclastogenesis and arthritis. Levels of IL-23p19 in joints of mice with CIA were determined. Lentiviral vectors expressing short hairpin RNA (shRNA) targeting IL-23p19 and lisofylline (LSF) were injected intraperitoneally into arthritic mice. Bone marrow-derived macrophages (BMMs) were treated with signal transducers and activators of transcription 4 (STAT4) specific shRNA and miR-223 sponge carried by lentiviral vectors in response to IL-23 stimulation. Treatment responses were determined by evaluating arthritis scores and histopathology in vivo, and detecting osteoclast differentiation and miR-223 levels in vitro. The binding of STAT4 to the promoter region of primary miR-223 (pri-miR-223) was determined in the Raw264.7 cell line. IL-23p19 expression was increased in the synovium of mice with CIA. Silencing IL-23p19 and inhibiting STAT4 activity ameliorates arthritis by reducing miR-223 expression. BMMs from mice in which STAT4 and miR-223 were silenced showed decreased osteoclast differentiation in response to IL-23 stimulation. IL-23 treatment increased the expression of miR-223 and enhanced the binding of STAT4 to the promoter of pri-miR-223. This study is the first to demonstrate that IL-23 promotes osteoclastogenesis by transcriptional regulation of miR-223 in murine macrophages and mice with CIA. Furthermore, our data indicate that LSF, a selective inhibitor of STAT4, should be an ideal therapeutic agent for treating RA through down-regulating miR-223-associated osteoclastogenesis.


Assuntos
Artrite Experimental , Artrite Reumatoide , MicroRNAs , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Camundongos , MicroRNAs/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , RNA Interferente Pequeno/metabolismo
5.
Mediators Inflamm ; 2022: 5255935, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091665

RESUMO

Objective: Activation of toll-like receptor 9 (TLR9) has been proposed to play an inhibitory role in RANKL-induced osteoclastogenesis. A20 deubiquitinase has been found to be related to bone loss. This study investigated the role of CpG oligodeoxynucleotides (CpG-ODNs) through regulation of A20 deubiquitinase in RANKL-induced osteoclast formation. Methods: RAW 264.7 cells, a murine monocyte-macrophage cell line, were incubated with or without CpG-ODN in the presence of RANKL. Osteoclast-specific genes and their related signaling molecules were measured by real-time quantitative polymerase chain reaction and Western blot assay. Morphological assessment for osteoclast formation was performed using tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring formation staining. Results: RANKL-induced osteoclast-related genes and proteins, c-Fos, NFATc1, TRAP, cathepsin K, and carbonic anhydrase II were significantly inhibited in RAW 264.7 cells stimulated with CpG-ODN. CpG-ODN attenuated TNF receptor-associated factor 6 (TRAF6), p-IκBα, and p-NF-κB expression in RAW 264 cells mediated by RANKL. CpG-ODN increased A20 gene and proteins in time-dependent manner. A20 expression under costimulation with CpG-ODN and RANKL was more decreased than under stimulation with RANKL alone. Cells transfected with A20 siRNA augmented expression of osteoclast-related genes and proteins. Number of TRAP-positive cells transfected with A20 siRNA was higher than those transfected with NC siRNA. A20 expression in cells transfected with IL-1ß siRNA in the presence of both RANKL and CpG-ODN was more decreased than those with NC siRNA. Conclusion: This study showed that CpG-ODN suppressed RANKL-induced osteoclast formation through regulation of the A20-TRAF6 axis in RAW 264.7 cells.


Assuntos
Osteoclastos , Fator 6 Associado a Receptor de TNF , Animais , Diferenciação Celular , Enzimas Desubiquitinantes/metabolismo , Camundongos , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Osteoclastos/metabolismo , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo
6.
Shanghai Kou Qiang Yi Xue ; 31(2): 113-119, 2022 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-36110065

RESUMO

PURPOSE: To investigate the correlation between the level of heat shock protein 90(Hsp90) and the amount of small extracellular vesicles(sEVs) in keratinocytes. METHODS: Human keratinocytes(HaCaT) were cultured in vivo and divided into wild-type group, short hairpin RNA interference group (shRNA group, low expression of Hsp90), and 17-Allylamino-17-demethoxygeldanamycin group (17-AAG group, Hsp90 protein inhibitor). sEVs were isolated from culture system by ultracentrifugation, and their morphological characteristics were observed under transmission electron microscopy (TEM). Western blotting was applied to identify the biological characteristics of sEVs. The number of sEVs particles was detected by nanoparticle tracking analysis (NTA). GraphPad Prism8.0 software was used to analyze the difference in the number of sEVs among the groups by t test (non-parametric Mann-Whitney U test). RESULTS: HaCaT-derived sEVs, obtained by ultracentrifugation, were consistent with the criteria of morphological and biological identification. No expression of Hsp90 protein was detected in HaCaT-derived sEVs. When interfered with Hsp90-shRNA, the number of sEVs were significantly increased. On day 5, the sEVs number of shRNA-interfering group was (177.4±4.18)×108(n=3), while that of vector group was (82.34±4.83)×108(n=3), and the difference was statistically significant (P<0.0001). After 5 days of inhibition with 17-AAG, the sEVs number of 17-AAG group was (652.5±26.73)×108(n=3) and that of control group was (262.22±5.44)×108(n=3), the difference was statistically significant (P<0.000 1). CONCLUSIONS: Low expression of Hsp90 protein can promote the secretion of sEVs in HaCaT cells. sEVs may be involved in the transfer of molecules between epithelial cells and immune cells.


Assuntos
Antineoplásicos , Vesículas Extracelulares , Antineoplásicos/farmacologia , Benzoquinonas , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Queratinócitos , Lactamas Macrocíclicas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
7.
Oxid Med Cell Longev ; 2022: 4674215, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36111165

RESUMO

Lipotoxicity can lead to beta-cell dysfunction and apoptosis because it induces oxidative stress. Recent studies have found that Irisin prevents pancreatic beta-cell dysfunction induced by palmitic acid (PA). However, an association between the protection against oxidative stress conferred by Irisin and beta-cell dysfunction has not been fully elucidated. In this study, we observed that Irisin treatment prevented INS-1 cell apoptosis induced by PA treatment and preserved the insulin-secreting function of INS-1 cells in vitro. These effects probably resulted from the Irisin-induced decrease in intracellular ROS levels triggered by PA treatment. In addition, PA treatment induced oxidative stress partially by inhibiting the activation of thioredoxin 2 (Trx2) through its increase of thioredoxin-interacting protein (Txnip) expression. However, Irisin administration blocked the increase in Txnip expression, which reversed the PA-induced inactivation of Trx2. Irisin also increased the nuclear translocation of Stat3, and the inhibition of Stat3 by siRNAs blocked Irisin-induced Trx2 expression, indicating that both Txnip and Stat3 are involved in Irisin-induced activation of Trx2. Furthermore, blockade of Stat3 by siRNAs led to the decreased gene expression of MafA and Ins and to cessation of glucose-induced insulin secretion that had been enhanced by Irisin. In vivo, HFD treatment led to reduced glucose tolerance and an increase in the level of the oxidative marker malondialdehyde (MDA) compared to that in the control group. However, these effects were ameliorated by Irisin injection due to the inhibition of beta-cell apoptosis and the activation of Trx2, probably through Txnip inhibition and Stat3 activation. In conclusion, our results reveal a possible mechanism for Irisin-induced beta-cell protection, which is mediated through Txnip inhibition and activation of the Stat3-Trx2 pathway.


Assuntos
Fibronectinas , Tiorredoxinas , Fibronectinas/metabolismo , Glucose/toxicidade , Insulina/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo , Ácido Palmítico/toxicidade , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Tiorredoxinas/metabolismo
8.
Cytokine ; 159: 156013, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36067712

RESUMO

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumours and has a high fatality rate worldwide. This study investigated the role of the Notch-1 signalling pathway in the pathogenesis and progression of GC. METHODS: A total of 64 patients with GC were included in this study. Immunohistochemistry staining was used to detect Notch-1 expression in tumour tissues and adjacent non-tumour tissues, and Notch-1 knockdown in GC cells was identified using short hairpin RNA. A cell scratch assay, transwell assay and flow cytometry analysis were used to analyse the effect of Notch-1 knockdown on cell proliferation, migration and cell cycle distribution. The expression of Notch-1, PTEN, Akt, ERK1/2, E-cadherin and other proteins was detected using Western blotting. RESULTS: The expression level of Notch-1 in GC tissues was higher than that in adjacent non-tumour tissues (P < 0.05). High levels of Notch-1 were also found to be associated with sex (male) and lymph node metastasis (P < 0.05). Notch-1 knockdown in the AGS and BGC-823 GC cell lines inhibited the migration and proliferation of GC cells, and Notch-1 knockdown arrested the cell cycle in the G0/G1 phase. PTEN protein expression was elevated in the presence of Notch-1 knockdown, resulting in the inhibition of phosphorylated Akt protein expression. In addition, phosphorylated ERK protein levels decreased in the presence of Notch-1 knockdown. Further inhibition of ERK1/2 signalling by the MEK1/2 inhibitor U0126 decreased the proliferation of AGS cells. The results of in vivo experiments with xenotransplantation in nude mice are consistent with these results. CONCLUSIONS: Notch-1 plays a key role in the development of GC and was found to promote the lymph node metastasis of GC. Notch-1 knockdown can effectively attenuate the progression of GC cells, which may function in part through the Notch-1-PTEN-ERK1/2 signalling axis.


Assuntos
Neoplasias Gástricas , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica , Metástase Linfática , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Nus , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Neoplasias Gástricas/patologia
9.
Cell Commun Signal ; 20(1): 144, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114543

RESUMO

BACKGROUND: Notch signaling is highly conserved and critically involved in cell differentiation, immunity, and survival. Activation of the Notch pathway modulates immune cell functions during the inflammatory response. However, it remains unknown whether and how the macrophage Notch1 may control the innate immune signaling TAK1, and RIPK3-mediated hepatocyte necroptosis in liver ischemia and reperfusion injury (IRI). This study investigated the molecular mechanisms of macrophage Notch1 in modulating TAK1-mediated innate immune responses and RIPK3 functions in liver IRI. METHODS: Myeloid-specific Notch1 knockout (Notch1M-KO) and floxed Notch1 (Notch1FL/FL) mice (n = 6/group) were subjected to 90 min partial liver warm ischemia followed by 6 h of reperfusion. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated ß-catenin knockout (KO) vector followed by LPS (100 ng/ml) stimulation. RESULTS: IR stress-induced Notch1 activation evidenced by increased nuclear Notch intracellular domain (NICD) expression in liver macrophages. Myeloid Notch1 deficiency exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil accumulation, and proinflammatory cytokines/chemokines production compared to the Notch1FL/FL controls. Unlike in the Notch1FL/FL controls, Notch1M-KO enhanced TRAF6, TAK1, NF-κB, RIPK3, and MLKL but reduced ß-catenin activation in ischemic livers. However, adoptive transfer of lentivirus ß-catenin-modified macrophages markedly improved liver function with reduced TRAF6, p-TAK1, RIPK3 and p-MLKL in IR-challenged livers. Moreover, disruption of RIPK3 in Notch1M-KO mice with an in vivo mannose-mediated RIPK3 siRNA delivery system diminished IR-triggered hepatocyte death. In vitro studies showed that macrophage NICD and ß-catenin co-localized in the nucleus, whereby ß-catenin interacted with NICD in response to LPS stimulation. Disruption of ß-catenin with a CRISPR/Cas9-mediated ß-catenin KO in Notch1FL/FL macrophage augmented TRAF6 activation leading to enhanced TAK1 function. While CRISPR/Cas9-mediated TRAF6 KO in Notch1M-KO macrophage inhibited RIPK3-mediated hepatocyte necroptosis after co-culture with primary hepatocytes. CONCLUSIONS: Macrophage Notch1 controls TAK1-mediated innate immune responses and RIPK3-mediated hepatocyte necroptosis through activation of ß-catenin. ß-catenin is required for the macrophage Notch1-mediated immune regulation in liver IRI. Our findings demonstrate that the macrophage Notch1-ß-catenin axis is a crucial regulatory mechanism in IR-triggered liver inflammation and provide novel therapeutic potential in organ IRI and transplant recipients. Video abstract.


Assuntos
Necroptose , Traumatismo por Reperfusão , Animais , Citocinas/metabolismo , Hepatócitos/metabolismo , Isquemia/metabolismo , Lipopolissacarídeos , Fígado/metabolismo , Macrófagos/metabolismo , Manose/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Traumatismo por Reperfusão/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , beta Catenina/metabolismo
10.
Fluids Barriers CNS ; 19(1): 73, 2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36076297

RESUMO

AIMS: To investigate whether DNA active demethylase TET regulates the expression of tight junction proteins in endothelial cells of the blood-brain barrier (BBB). METHODS: Correlations between TET2 activity (indicated by its catalytic product 5hmC) and the expression of BBB tight junction proteins were examined in Tet2 knockout mice and post-mortem human brain tissues. In cultured endothelial cells, the impact of changes of TET activity on the expression of tight junction protein, ZO-1, was studied. BBB permeability assays were performed in Tet2 knockout mice. RESULTS: It was found that the level of 5hmC decreased in brain microvascular endothelial cells of aging mice. In Tet2 knockout mice, the level of 5hmC in endothelial cells was weaker and significantly correlated with the reduced expression of tight junction protein ZO-1. In cultured endothelial cells, H2O2 significantly decreased the expression of 5hmC and ZO-1. Tet2 knock-down using siRNA significantly downregulated the expression of ZO-1 in endothelial cells. hMeChip-PCR showed that H2O2 decreased the level of 5hmC in the ZO-1 promoter region, which was rescued by N-acetyl cysteine (NAC). Consistently, Tet2 knock-down using siRNA significantly downregulated the level of 5hmC in the ZO-1 promoter region. It was also found that the level of 5hmC decreased in endothelial cells of aging human brains compared with that of adult brains, and the level of ZO-1 was positively correlated with that of 5hmC in microvascular endothelial cells. CONCLUSIONS: These findings suggest that TET activity is essential in regulating ZO-1 expression of BBB. It might be a potential target for neuroprotection during aging and in diverse neurological conditions.


Assuntos
Dioxigenases , Células Endoteliais , Animais , Barreira Hematoencefálica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Células Endoteliais/metabolismo , Epigênese Genética , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
11.
Can Respir J ; 2022: 8437348, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091328

RESUMO

Introduction: Vascular smooth muscle cells (VSMCs) are highly involved in airway vascular remodeling in asthma. Objectives: This study aimed to investigate the mechanisms underlying the effects of a disintegrin and metalloproteinase-33 (ADAM33) gene on the migration capacity and inflammatory cytokine secretion of VSMCs. Methods: Human aortic smooth muscle cells (HASMCs) were transfected with lentiviral vectors carrying short hairpin RNA (shRNA) targeting ADAM33 or negative control vectors. The migration capacity of HASMCs was evaluated by a transwell assay. The levels of secreted inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits. Reverse transcription-quantitative polymerase chain reaction and Western blot assays were performed to detect mRNA and protein expression levels. Results: Silencing of ADAM33 significantly inhibited the migration of HASMCs. The expression of tumor necrosis factor alpha (TNF-α) in the supernatant of HASMCs was decreased, while that of interferon gamma (IFN-γ) was increased after the transfection of shRNA targeting ADAM33. Insufficient ADAM33 expression also suppressed the expression levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (AKT), phospho-mammalian target of rapamycin (mTOR), Rho-associated protein kinases, phospho-forkhead box protein O1 (FOXO1), and cyclin D1, but it did not affect the levels of AKT, mTOR, or Rho. Conclusion: Silencing of the ADAM33 gene inhibited HASMC migration and regulated inflammatory cytokine secretion via targeting the PI3K/AKT/mTOR pathway and its downstream signaling. These data contribute to a better understanding of the regulatory mechanisms of airway vascular remodeling in asthma.


Assuntos
Asma , Proteínas Proto-Oncogênicas c-akt , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas ADAM/farmacologia , Remodelação das Vias Aéreas , Asma/genética , Asma/metabolismo , Movimento Celular , Células Cultivadas , Citocinas , Humanos , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinase/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia , Remodelação Vascular
12.
Int J Mol Sci ; 23(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36077317

RESUMO

A prolonged pandemic with numerous human casualties requires a rapid search for means to control the various strains of SARS-CoV-2. Since only part of the human population is affected by coronaviruses, there are probably endogenous compounds preventing the spread of these viral pathogens. It has been shown that piRNA (PIWI-interacting RNAs) interact with the mRNA of human genes and can block protein synthesis at the stage of translation. Estimated the effects of piRNA on SARS-CoV-2 genomic RNA (gRNA) in silico. A cluster of 13 piRNA binding sites (BS) in the SARS-CoV-2 gRNA region encoding the oligopeptide was identified. The second cluster of BSs 39 piRNAs also encodes the oligopeptide. The third cluster of 24 piRNA BS encodes the oligopeptide. Twelve piRNAs were identified that strongly interact with the gRNA. Based on the identified functionally important endogenous piRNAs, synthetic piRNAs (spiRNAs) are proposed that will suppress the multiplication of the coronavirus even more strongly. These spiRNAs and selected endogenous piRNAs have little effect on human 17494 protein-coding genes, indicating a low probability of side effects. The piRNA and spiRNA selection methodology created for the control of SARS-CoV-2 (NC_045512.2) can be used to control all strains of SARS-CoV-2.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/genética , Genoma , Humanos , RNA Guia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/genética
13.
Cells ; 11(17)2022 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-36078084

RESUMO

The C-type lectin receptors (CLRs) Dectin-1 and Dectin-2 are involved in several innate immune responses and are expressed mainly in dendritic cells, monocytes, and macrophages. Dectin-1 activation exacerbates obesity, inflammation, and insulin resistance/type 2 diabetes (T2D). However, the role of Dectin-2 is not clear in T2D. This study aims to evaluate the expression and function of Dectin-2 in peripheral blood mononuclear cells (PBMCs) isolated from diabetic patients and non-diabetic controls. Flow-cytometry and qRT-PCR were performed to evaluate the expression of Dectin-2 in different leukocyte subpopulations isolated from T2D patients (n = 10) and matched non-diabetic controls (n = 11). The functional activity of Dectin-2 was identified in PBMCs. CRP, IL-1ß, and TNF-α concentrations were determined by ELISA. siRNA transfection and Western blotting were performed to assess p-Syk and p-NF-kB expression. siRNA transfection was performed to knock down the gene of interest. Our results show that Dectin-2 expression was the highest in monocytes compared with other leukocyte subpopulations. The expression of Dectin-2 was significantly increased in the monocytes of T2D patients compared with non-diabetic controls. Dectin-2 expression positively correlated with markers of glucose homeostasis, including HOMA-IR and HbA1c. The expression of inflammatory markers was elevated in the PBMCs of T2D patients. Interestingly, SOCS3, a negative regulator of inflammation, was expressed significantly lowlier in the PBMCs of T2D patients. Moreover, SOCS3 expression was negatively correlated with Dectin-2 expression level. The further analysis of inflammatory signaling pathways showed a persistent activation of the Dectin-2-Syk-NFkB pathway that was instigated by the diminished expression of SOCS3. Dectin-2 activation failed to induce SOCS3 expression and suppress subsequent inflammatory responses in the PBMCs of diabetic patients. siRNA-mediated knockdown of SOCS3 in PBMCs displayed a similar inflammatory phenotype to diabetic PBMCs when exposed to Dectin-2 ligands. Altogether, our findings suggest that elevated Dectin-2 and its relationship with SOCS3 could be involved in the abnormal immune response observed in T2D patients.


Assuntos
Diabetes Mellitus Tipo 2 , Leucócitos Mononucleares , Biomarcadores/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Inflamação/metabolismo , Lectinas Tipo C , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
14.
FASEB J ; 36(10): e22526, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36063123

RESUMO

The (Pro)renin receptor (PRR) is reportedly involved in hepatic lipid metabolism and hepatocyte PRR knockdown protects mice against hepatosteatosis. However, the impact of PRR inhibition on liver inflammation and fibrosis in nonalcoholic steatohepatitis (NASH) remains unclear. Herein, C57BL/6 mice were fed a normal chow diet or fast food diet (FFD) for 24 weeks. Lentivirus-mediated PRR short hairpin RNA (shRNA) or handle region peptide (HRP), a PRR blocker, was administered for PRR inhibition. Mouse primary hepatocytes were cultured with palmitic acid, prorenin, siRNA-targeted PRR, and HRP. In FFD-fed mice, PRR inhibition via lentivirus-mediated PRR knockdown or HRP significantly attenuated liver steatosis, inflammation, and fibrosis. Mechanistically, PRR knockdown or HRP decreased hepatic acetyl-CoA carboxylase (ACC) abundance and upregulated peroxisome proliferator-activated receptor-alpha (PPARα). HRP treatment also decreased hepatic PRR expression. In addition, intrahepatic oxidative stress, apoptosis and inflammatory cell recruitment were ameliorated by PRR knockdown or HRP treatment, along with suppression of proinflammatory cytokine expression. PRR inhibition downregulated the hepatic expression of profibrotic factors, as well as TGF-ß1/SMAD3 pathway. In primary mouse hepatocytes, PRR knockdown with siRNA or HRP downregulated cellular ACC and increased PPARα expression. In conclusion, our findings revealed that PRR inhibition attenuated hepatic steatosis, inflammation, and fibrosis in mice with NASH. Accordingly, targeting PRR signaling may serve as a potential treatment for NASH.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Modelos Animais de Doenças , Fibrose , Inflamação/metabolismo , Fígado/metabolismo , Cirrose Hepática/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , RNA Interferente Pequeno/metabolismo , Renina/metabolismo
15.
FASEB J ; 36(10): e22542, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36094000

RESUMO

Ischemic stroke is one of the major causes of morbidity and mortality. The ß-1, 3-galactosyltransferase 2 (B3galt2), a member of ß-1, 3-galactosyltransferase family, is playing a vital role in the pathological process of cerebral ischemic injury, but its underlying mechanisms remain unclear. In the present study, we examined the involvement of oxidative stress and NLRP3 inflammasome activation in the neuroprotective effect of B3galt2. Cerebral ischemia/reperfusion (I/R) injury was simulated in a mouse middle cerebral artery occlusion (MCAO) model. Recombinant human B3galt2 (rh-B3galt2) was administered intranasally 1 h post MCAO, and TGF-ß1-siRNA was administered intracerebroventricularly 24 h before MCAO. Outcome measures included brain infarct volume, neurological function, blood-brain barrier (BBB) permeability, neuronal apoptosis, oxidative stress, and the inflammatory response. First, we found that rh-B3galt2 significantly alleviated brain infarct volume and BBB permeability, improved neurological function, and attenuated I/R-induced neuron apoptosis and oxidative stress. Furthermore, rh-B3galt2 attenuated pro-inflammatory cytokines, NF-κB, IL-6, TNF-α, and IL-1ß, and inhibited NLRP3 inflammasome activation. Finally, inhibition of TGF-ß1 by TGF-ß1-siRNA abolished the anti-oxidative and anti-inflammatory effects of rh-B3galt2 in mice after I/R. Collectively, our study demonstrated that rh-B3galt2 exerts neuroprotective effects by regulating cerebral ischemia-induced oxidative stress and NLRP3 inflammasome, which is mainly dependent on the heightening of the TGF-ß1 pathway. Thus, B3galt2 might be considered a new therapeutic target for ischemic stroke treatment.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Administração Intranasal , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologia , Galactosiltransferases/uso terapêutico , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
16.
Nan Fang Yi Ke Da Xue Xue Bao ; 42(8): 1126-1133, 2022 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-36073210

RESUMO

OBJECTIVE: To investigate the effect of interference of CTPS gene on toosendanin-induced apoptosis of gastric cancer MKN-45 cells. METHODS: Bioinformatic analysis was used to analyze CTPS gene expression in human gastric cancer tissues and the overall survival of gastric cancer patients with high CTPS gene expression. Human gastric cancer MKN-45 cells were transfected with a short hairpin interfering RNA targeting CTPS gene, and 48 h later, qRT-PCR and Western blotting were used to detect cellular expression CTPS at both the mRNA and protein levels. MKN-45 cells with CTPS knockdown were treated with 80 nmol/L toosendanin for 48 h, and the cell viability was assessed with MTT assay; the cell morphology was observed using laser confocal microscope, and the expression of γH2AX was detected with immunofluorescence assay. RESULTS: Bioinformatic analysis suggested that CTPS was highly expressed in human gastric cancer tissues, and gastric cancer patients with high CTPS gene expression had a shorter overall survival. MKN-45 cells transfected with Sh-CTPS interference vector showed significantly lowered cell survival rate (P < 0.01) with obvious cell shrinkage, irregular morphology, typical apoptotic changes, and increased cell apoptosis rate (P < 0.05). Treatment of the transfected cells with 80 nmol/L toosendanin for 48 h resulted in further reduction of the cell survival rate (P < 0.001), and the cells showed an increased apoptotic rate (P < 0.05) with appearance of apoptotic bodies. CONCLUSION: Interference of CTPS gene can promote TSN-induced apoptosis of gastric cancer MKN-45 cells.


Assuntos
Neoplasias Gástricas , Apoptose , Linhagem Celular Tumoral , Humanos , RNA Interferente Pequeno/metabolismo , Silanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Triterpenos
17.
Int J Biol Sci ; 18(13): 5154-5167, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35982907

RESUMO

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets (GSE28829, GSE43292, GSE100927, and GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.


Assuntos
Aterosclerose , Fosfoenolpiruvato Carboxiquinase (ATP) , Placa Aterosclerótica , Animais , Aterosclerose/metabolismo , Movimento Celular , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/genética , Neointima/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo
18.
Cell Rep ; 40(8): 111263, 2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36001962

RESUMO

In animal germlines, transposons are silenced at the transcriptional or post-transcriptional level to prevent deleterious expression. Ciliates employ a more direct approach by physically eliminating transposons from their soma, utilizing piRNAs to recognize transposons and imprecisely excise them. Ancient, mutated transposons often do not require piRNAs and are precisely eliminated. Here, we characterize the Polycomb Repressive Complex 2 (PRC2) in Paramecium and demonstrate its involvement in the removal of transposons and transposon-derived DNA. Our results reveal a striking difference between the elimination of new and ancient transposons at the chromatin level and show that the complex may be guided by Piwi-bound small RNAs (sRNAs). We propose that imprecise elimination in ciliates originates from an ancient transposon silencing mechanism, much like in plants and metazoans, through sRNAs, repressive methylation marks, and heterochromatin formation. However, it is taken a step further by eliminating DNA as an extreme form of transposon silencing.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , DNA/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Inativação Gênica , Complexo Repressor Polycomb 2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
19.
Cells ; 11(16)2022 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-36010574

RESUMO

Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone-marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes.


Assuntos
Ativação de Macrófagos , Receptor 4 Toll-Like , Animais , Biomarcadores/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , Fenótipo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
20.
Stem Cell Res Ther ; 13(1): 397, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927735

RESUMO

BACKGROUND: Diabetes-related osteoporosis (DOP) is a chronic disease caused by the high glucose environment that induces a metabolic disorder of osteocytes and osteoblast-associated mesenchymal stem cells. The processes of bone defect repair and regeneration become extremely difficult with DOP. Adipose-derived stem cells (ASCs), as seed cells in bone tissue engineering technology, provide a promising therapeutic approach for bone regeneration in DOP patients. The osteogenic ability of ASCs is lower in a DOP model than that of control ASCs. DNA methylation, as a mechanism of epigenetic regulation, may be involved in DNA methylation of various genes, thereby participating in biological behaviors of various cells. Emerging evidence suggests that increased DNA methylation levels are associated with activation of Wnt/ß-catenin signaling pathway. The purpose of this study was to investigate the influence of the diabetic environment on the osteogenic potential of ASCs, to explore the role of DNA methylation on osteogenic differentiation of DOP-ASCs via Wnt/ß-catenin signaling pathway, and to improve the osteogenic differentiation ability of ASCs with DOP. METHODS: DOP-ASCs and control ASCs were isolated from DOP C57BL/6 and control mice, respectively. The multipotency of DOP-ASCs was confirmed by Alizarin Red-S, Oil Red-O, and Alcian blue staining. Real-time polymerase chain reaction (RT-PCR), immunofluorescence, and western blotting were used to analyze changes in markers of osteogenic differentiation, DNA methylation, and Wnt/ß-catenin signaling. Alizarin Red-S staining was also used to confirm changes in the osteogenic ability. DNMT small interfering RNA (siRNA), shRNA-Dnmt3a, and LVRNA-Dnmt3a were used to assess the role of Dnmt3a in osteogenic differentiation of control ASCs and DOP-ASCs. Micro-computed tomography, hematoxylin and eosin staining, and Masson staining were used to analyze changes in the osteogenic capability while downregulating Dnmt3a with lentivirus in DOP mice in vivo. RESULTS: The proliferative ability of DOP-ASCs was lower than that of control ASCs. DOP-ASCs showed a decrease in osteogenic differentiation capacity, lower Wnt/ß-catenin signaling pathway activity, and a higher level of Dnmt3a than control ASCs. When Dnmt3a was downregulated by siRNA and shRNA, osteogenic-related factors Runt-related transcription factor 2 and osteopontin, and activity of Wnt/ß-catenin signaling pathway were increased, which rescued the poor osteogenic potential of DOP-ASCs. When Dnmt3a was upregulated by LVRNA-Dnmt3a, the osteogenic ability was inhibited. The same results were obtained in vivo. CONCLUSIONS: Dnmt3a silencing rescues the negative effects of DOP on ASCs and provides a possible approach for bone tissue regeneration in patients with diabetic osteoporosis.


Assuntos
Diabetes Mellitus , Osteoporose , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , DNA/metabolismo , DNA Metiltransferase 3A , Diabetes Mellitus/genética , Regulação para Baixo , Epigênese Genética , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese , Osteoporose/tratamento farmacológico , Osteoporose/terapia , RNA Interferente Pequeno/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética , Microtomografia por Raio-X , beta Catenina/genética , beta Catenina/metabolismo
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