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1.
Proc Natl Acad Sci U S A ; 120(3): e2209184120, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36626553

RESUMO

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.


Assuntos
Monócitos , NADPH Oxidases , Quinases Associadas a rho , Humanos , Aminoácidos , Monócitos/metabolismo , NADPH Oxidases/metabolismo , Fosfoproteínas/metabolismo , Espécies Reativas de Oxigênio , Quinases Associadas a rho/metabolismo
2.
Funct Integr Genomics ; 23(1): 43, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658407

RESUMO

MicroRNA (miR)-381-3p is the newly discovered tumor-associated miRNA, which is frequently associated with diverse human malignancies; but, it is still unknown about its effect on acute myeloid leukemia (AML) in children. This work focused on exploring miR-381-3p's effect on childhood AML and identifying the possible mechanisms facilitating new treatment development. Using qRT-PCR analysis, miR-381-3p expression remarkably reduced in pediatric AML patients and AML cell lines (HL-60 and U937). Following transfection of miR-381-3p mimic or inhibitor into HL-60 and U937 cells, we conducted MTT assay to evaluate cell proliferation, flow cytometry (FCM) to measured cell apoptosis and cell cycle, whereas Transwell assays to detect cell invasion and migration. Our results demonstrated that miR-381-3p overexpression remarkably repressed cell growth, invasion and migration; additionally, miR-381-3p overexpression resulted in arrest of cell cycle and enhanced cell apoptosis. In contrast, miR-381-3p knockdown led to an opposite effect. Moreover, we predicted miR-381's target gene and validated it by luciferase reporter assay and TargetScan, separately. We identified miR-381-3p's binding site in ROCK1 3'-UTR. As revealed by Western-blot (WB) assay, miR-381-3p overexpression notably suppressed ROCK1 level. Moreover, restoring ROCK1 expression abolished miR-381-3p's inhibition on cell proliferation, invasion and migration. Data in this work indicated the role of miR-381-3p as the tumor suppressor within pediatric AML by targeting ROCK1. Therefore, miR-381-3p might serve as a potential therapeutic target for the treatment of pediatric AML.


Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Humanos , Criança , Regulação para Baixo , MicroRNAs/genética , MicroRNAs/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Linhagem Celular , Proliferação de Células/genética , Linhagem Celular Tumoral , Apoptose/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
3.
Cells ; 12(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36672200

RESUMO

The reaction field of abnormal vascular contraction induced by sphingosylphosphorylcholine (SPC) and the action point of SPC around the plasma membranes remain unknown. However, we found in a previous study that fisetin prevents SPC-induced vascular smooth muscle cells contraction, while the mechanism remains unknown. Therefore, in this study, we aimed to address the action point of SPC around the plasma membranes and the involvement of fisetin. We focused on microdomains and evaluated their markers flotillin-1 and caveolin-1 and the localization of SPC to investigate their action point. The results showed that microdomains of vascular smooth muscle cells were not involved in SPC-induced contraction. However, we found that after SPC had been affected on the plasma membrane, cells took up SPC via endocytosis. Moreover, SPC remained in the cells and did not undergo transcytosis, and SPC-induced contracting cells produced exosomes. These phenomena were similar to those observed in fisetin-treated cells. Thus, we speculated that, although not involved in the reaction field of SPC-induced contractions, the microdomain induced the endocytosis of SPCs, and fisetin prevented the contractions by directly targeting vascular smooth muscle cells. Notably, this preventive mechanism involves the cellular uptake of SPC via endocytosis.


Assuntos
Músculo Liso Vascular , Quinases Associadas a rho , Músculo Liso Vascular/metabolismo , Quinases Associadas a rho/metabolismo , Contração Muscular/fisiologia , Endocitose
4.
PLoS One ; 18(1): e0270288, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36719899

RESUMO

BACKGROUND: The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells. METHODS: Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10µM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses. RESULTS: Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ. CONCLUSION: ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.


Assuntos
Transdução de Sinais , Quinases Associadas a rho , Camundongos , Humanos , Animais , Quinases Associadas a rho/metabolismo , Células Estreladas do Fígado/metabolismo , Células Cultivadas
5.
Eur J Pharmacol ; 940: 175465, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36566915

RESUMO

Liver cancer is a kind of malignant tumor with poor sensitivity to chemotherapy. It is urgent to investigate approaches to improve the outcome of chemotherapy. KDM5A has been reported to be an oncogene in various cancers and is associated with drug resistance. However, the functions of KDM5A in chemotherapeutic sensitivity of liver cancer not been well illustrated. In this study, we found that KDM5A was upregulated in liver cancer tissue and cell lines. KDM5A knockdown using a gene interference strategy suppressed the growth of liver cancer in vitro and in vivo. CPI-455, a pharmacological inactivation of KDM5A enhanced the cytotoxicity of cisplatin (CDDP) in liver cells. CPI-455 and CDDP cotreatment resulted in apoptosis and mitochondrial dysfunction. We also found that knockdown or inactivation of KDM5A resulted in the downregulation of ROCK1, an oncogene regulating the activation of the PTEN/AKT signaling pathway. In particular, overexpression of ROCK1 or SF1670, a pharmacological inhibitor of PTEN, alleviated the cytotoxicity of CPI-455 and CDDP cotreatment. In HCCLM3 xenografts, CPI-455 and CDDP cotreatment dramatically inhibited the growth of xenograft tumor compared to CPI-455 or CDDP treatment alone. In conclusion, this study suggested that targeting the inactivation of KDM5A is an efficient strategy to enhance the chemosensitivity of liver cancer cells to CDDP by modulating the ROCK1/PTEN/AKT signaling pathway.


Assuntos
Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Transdução de Sinais , Apoptose , Neoplasias Hepáticas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Quinases Associadas a rho/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
6.
J Appl Physiol (1985) ; 134(1): 152-159, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36519569

RESUMO

The time course of smooth muscle contraction can be divided into two phases, the initial phase is associated with force development, whereas the sustained phase is associated with force maintenance. Cumulative evidence suggests that the two phases are regulated by different signaling pathways and that ρ-kinase (ROCK) and protein kinase C (PKC) play an important role in regulating isometric force in sustained contractions. Since the maintenance of sustained force is critical to the function of vascular smooth muscle, unraveling the complex mechanism of force maintenance is crucial for understanding the cell biology of the muscle. The present study examined the effects of ROCK and PKC on the level of phosphorylation of the 20-kD myosin light chain (MLC20) and isometric force during a sustained contraction. We used partial activation and inhibition of ROCK and PKC to reduce the isometric force by 50% of the maximal isometric force in fully activated muscle, Fmax. We then examined the level of MLC20 phosphorylation in each case. We found that in partially activated muscle the level of MLC20 phosphorylation required to maintain 50% Fmax was much lower than that required in muscles where 50% reduction in Fmax was achieved by partial inhibition of ROCK and PKC. The results can be explained by a model containing a contractile apparatus and a cytoskeletal scaffold where force generated by the contractile apparatus is transmitted to the extracellular domain through the cytoskeleton. The results indicate that ROCK and PKC play an important role in force transmission through the cytoskeleton.NEW & NOTEWORTHY The study supports a model that the maintenance of sustained force during a contraction of arterial smooth muscle is dependent on the intracellular transmission of force through the cytoskeleton and that ρ-kinase and protein kinase C plays an important role in the regulation of cytoskeletal integrity and its efficiency in force transmission.


Assuntos
Proteína Quinase C , Quinases Associadas a rho , Animais , Ovinos , Proteína Quinase C/metabolismo , Quinases Associadas a rho/metabolismo , Contração Muscular/fisiologia , Músculo Liso Vascular/metabolismo , Artérias Carótidas/metabolismo , Fosforilação
7.
Shock ; 59(1): 99-107, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36476974

RESUMO

ABSTRACT: Background: Acute lung injury (ALI) induced by sepsis is distinguished by an inflammatory progression. Herein, we investigated the action of circular RNA kelch like family member 2 (circKlhl2) in sepsis-induced ALI. Methods: The animal or cell model of sepsis ALI was established by LPS stimulation. The contents of circKlhl2, microRNA-29b-3p (miR-29b-3p), rho-associated coiled-coil containing protein kinase 1 (ROCK1), CyclinD1, B-cell lymphoma-2 (Bcl-2), and cleaved-caspase 3 (C-caspase-3) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. Cell viability was assessed by cell counting kit 8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. The abundances of proinflammatory cytokines were detected by enzyme-linked immunosorbent assay. Besides, the targeted relationship between miR-29b-3p and circKlhl2 or ROCK1 was verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Results: Loss of circKlhl2 mitigated lung injury and proinflammatory cytokine expression in sepsis-ALI mice model and alleviated LPS-induced apoptosis and inflammatory response in microvascular endothelial cell (MPVECs) in vitro . The abundances of circKlhl2 and ROCK1 were boosted, while the miR-29b-3p level was diminished in the animal or cell model of sepsis-ALI. MiR-29b-3p inhibition abrogated circKlhl2 knockdown-mediated effects on MPVECs injury. Moreover, miR-29b-3p overexpression promoted cell proliferation and inhibited apoptosis and inflammation in LPS-treated MPVECs, while ROCK1 enhancement reversed these effects. Conclusion: CircKlhl2 expedited the sepsis-induced ALI by adjusting miR-29b-3p/ROCK1 axis.


Assuntos
Lesão Pulmonar Aguda , MicroRNAs , Sepse , Camundongos , Animais , Regulação para Baixo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Lesão Pulmonar Aguda/metabolismo , Sepse/complicações , Sepse/genética , Apoptose/genética
8.
Respir Res ; 23(1): 380, 2022 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-36575527

RESUMO

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive disorder that causes airway obstruction and lung inflammation. The first-line treatment of COPD is the bronchodilators of ß2-agonists and antimuscarinic drugs, which can help control the airway obstruction, but the long-term use might render the drug tolerance. Bisphosphonates are widely used in osteoclast-mediated bone diseases treatment for decades. For drug repurposing, can delivery of a third generation of nitrogen-containing bisphosphonate, risedronate (RIS) ameliorate the progression of COPD? METHODS: COPD rats or mice models have been established through cigarette-smoking and elastase injection, and then the animals are received RIS treatment via nebulization. Lung deposition of RIS was primarily assessed by high-performance liquid chromatography (HPLC). The respiratory parameters of airway obstruction in COPD rats and mice were documented using plethysmography method and resistance-compliance system. RESULTS: High lung deposition and bioavailability of RIS was monitored with 88.8% of RIS input dose. We found that RIS could rescue the lung function decline of airspace enlargement and mean linear intercept in the COPD lung. RIS could curb the airway obstruction by suppressing 60% of the respiratory resistance and elevating the airway's dynamic compliance, tidal volume and mid-expiratory flow. As an inhibitor of farnesyl diphosphate synthase (FDPS), RIS suppresses FDPS-mediated RAS and RhoA prenylation to obstruct its membrane localization in airway smooth muscle cells (ASMCs), leading to the inhibition of downstream ERK-MLCK and ROCK1-MLCP pathway to cause ASMCs relaxation. Additionally, RIS nebulization impeded pro-inflammatory cell accumulation, particularly macrophages infiltration in alveolar parenchyma. The NF-κB, tumor necrosis factor-alpha, IL-1ß, IL-8, and IL-6 declined in microphages following RIS nebulization. Surprisingly, nebulization of RIS could overcome the tolerance of ß2-agonists in COPD-rats by increasing the expression of ß2 receptors. CONCLUSIONS: Nebulization of RIS could alleviate airway obstruction and lung inflammation in COPD, providing a novel strategy for treating COPD patients, even those with ß2-agonists tolerance.


Assuntos
Obstrução das Vias Respiratórias , Doença Pulmonar Obstrutiva Crônica , Ratos , Camundongos , Animais , NF-kappa B/metabolismo , Ácido Risedrônico/uso terapêutico , Pulmão/metabolismo , Inflamação/metabolismo , Prenilação , Quinases Associadas a rho/metabolismo
9.
Molecules ; 27(23)2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36500403

RESUMO

Ginsenoside Rh1 (G-Rh1), a possible bioactive substance isolated from the Korean Panax ginseng Meyer, has a wide range of pharmacological effects. In this study, we have investigated the anticancer efficacy of G-Rh1 via in silico and in vitro methodologies. This study mainly focuses on the two metastatic regulators, Rho-associated protein kinase 1 (ROCK1) and RhoA, along with other standard apoptosis regulators. The ROCK1 protein is a member of the active serine/threonine kinase family that is crucial for many biological processes, including cell division, differentiation, and death, as well as many cellular processes and muscle contraction. The abnormal activation of ROCK1 kinase causes several disorders, whereas numerous studies have also shown that RhoA is expressed highly in various cancers, including colon, lung, ovarian, gastric, and liver malignancies. Hence, inhibiting both ROCK1 and RhoA will be promising in preventing metastasis. Therefore, the molecular level interaction of G-Rh1 with the ROCK1 and RhoA active site residues from the preliminary screening clearly shows its inhibitory potential. Molecular dynamics simulation and principal component analysis give essential insights for comprehending the conformational changes that result from G-Rh1 binding to ROCK1 and RhoA. Further, MTT assay was employed to examine the potential cytotoxicity in vitro against human lung cancer cells (A549) and Raw 264.7 Murine macrophage cells. Thus, G-Rh1 showed significant cytotoxicity against human lung adenocarcinoma (A549) at 100 µg/mL. In addition, we observed an elevated level of reactive oxygen species (ROS) generation, perhaps promoting cancer cell toxicity. Additionally, G-Rh1 suppressed the mRNA expression of RhoA, ROCK1, MMP1, and MMP9 in cancer cell. Accordingly, G-Rh1 upregulated the p53, Bax, Caspase 3, caspase 9 while Bcl2 is downregulated intrinsic pathway. The findings from our study propose that the anticancer activity of G-Rh1 may be related to the induction of apoptosis by the RhoA/ROCK1 signaling pathway. As a result, this study evaluated the functional drug-like compound G-Rh1 from Panax ginseng in preventing and treating lung cancer adenocarcinoma via regulating metastasis and apoptosis.


Assuntos
Ginsenosídeos , Neoplasias Pulmonares , Panax , Humanos , Camundongos , Animais , Células A549 , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Ginsenosídeos/química , Apoptose , Panax/metabolismo
10.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 34(12): 1268-1272, 2022 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-36567581

RESUMO

OBJECTIVE: To explore the effect of Rho kinase inhibitor on intestinal injury in septic rats and its possible mechanism. METHODS: Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), Rho kinase inhibitor Y-27632 control group (Y+Sham group), sepsis model group [cecal ligation and puncture (CLP) group] and Y-27632 pretreatment group (Y+CLP group), with 8 rats in each group. Rat sepsis model was reproduced by CLP. The rats in the Sham group and Y+Sham group were only separated and moved the cecum without ligation and perforation. The rats in the Y+Sham group and Y+CLP group were pretreated with intraperitoneal injection of Y-27632 solution 5 mg/kg 15 minutes before operation; the rats in the Sham group and CLP group were intraperitoneally injected with the same amount of phosphate buffered saline (PBS). Twenty-four hours after operation, the heart blood was collected and the serum diamine oxidase (DAO) content was determined by enzyme-linked immunosorbent assay (ELISA). Then the small intestine tissue was collected, the pathological changes of the intestinal tissue were observed under the light microscope after hematoxylin-eosin (HE) staining, and Chiu's score was performed. The positive expressions of Rho-related coiled-coil kinase 1 (ROCK1) and nuclear factor-κB (NF-κB) in intestinal tissue were detected by immunohistochemistry. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in intestinal tissue homogenate. RESULTS: The intestinal tissue structure of the Sham group and Y+Sham group was intact and the mucosa was arranged neatly. Compared with the Sham group, the intestinal mucosa of the CLP group was arranged disorderly, with a large number of inflammatory cells infiltration, and the Chiu's score was significantly increased (3.83±0.27 vs. 0.12±0.11, P < 0.05), indicating that those rats suffered from septic intestinal injury. Compared with the CLP group, the degree of necrosis of intestinal epithelial cells in the Y+CLP group was reduced, a small amount of inflammatory cells infiltration was seen, and the Chiu's score was significantly decreased (2.85±0.21 vs. 3.83±0.27, P < 0.05), indicating that Y-27632 pretreatment could alleviate intestinal injury in septic rats. Compared with the Sham group, the positive expressions of intestinal tissue ROCK1 and NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the CLP group were significantly increased [ROCK1 expression (A value): 0.19 (0.18, 0.22) vs. 0.10 (0.09, 0.11), NF-κB expression (A value): 0.40±0.02 vs. 0.15±0.01, DAO (ng/L): 287.81±23.31 vs. 144.92±17.72, TNF-α (ng/L): 101.08±5.62 vs. 74.81±5.56, all P < 0.05], the level of intestinal homogenate IL-10 was significantly decreased (µg/L: 55.16±5.20 vs. 95.95±7.53, P < 0.05). Compared with the CLP group, the positive expressions of intestinal tissue ROCK1, NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the Y+CLP group were significantly decreased [ROCK1 expression (A value): 0.15 (0.13, 0.18) vs. 0.19 (0.18, 0.22), NF-κB expression (A value): 0.28±0.01 vs. 0.40±0.02, DAO (ng/L): 243.34±19.76 vs. 287.81±23.31, TNF-α (ng/L): 90.41±8.79 vs. 101.08±5.62, all P < 0.05], while the level of intestinal homogenate IL-10 was significantly increased (µg/L: 66.15±5.74 vs. 55.16±5.20, P < 0.05), indicating that the protective effect of Y-27632 pretreatment on sepsis intestinal injury rats might be related to the regulation of RhoA/ROCK1/NF-κB signaling pathway. CONCLUSIONS: Rho kinase inhibitors can reduce intestinal injury in septic rats, and the mechanism may be related to inhibiting RhoA/ROCK1/NF-κB signaling pathway and reducing intestinal inflammation in septic rats.


Assuntos
Enterite , NF-kappa B , Sepse , Animais , Masculino , Ratos , Interleucina-10 , NF-kappa B/metabolismo , Ratos Sprague-Dawley , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Sepse/complicações , Sepse/tratamento farmacológico , Sepse/metabolismo , Fator de Necrose Tumoral alfa , Enterite/tratamento farmacológico , Enterite/etiologia , Enterite/metabolismo
11.
Cells ; 11(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36359898

RESUMO

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases. Increasing evidence indicates that obesity, itself being a low-grade inflammatory disease, predisposes to a variety of other chronic inflammatory diseases. Previous studies indicated that this later might be partially related to an impaired efferocytosis induced by increased uptake of circulating saturated fatty acids by macrophages in obese people. Here, we show that palmitate inhibits efferocytosis by bone marrow-derived macrophages in a dose-dependent manner. Palmitate triggers autophagy but also activates an energy-sensing mTORC1/ROCK1 signaling pathway, which interferes with the autophagosome-lysosome fusion, resulting in accumulation of the cellular membranes in autophagosomes. We propose that lack of sufficient plasma membrane supply attenuates efferocytosis of palmitate-exposed macrophages. AMP-activated protein kinase activators lead to mTORC1 inhibition and, consequently, released the palmitate-induced efferocytosis block in macrophages. Thus, they might be useful in the treatment of obesity not only by affecting metabolism thought so far. ROCK1 inhibitors could also be considered.


Assuntos
Palmitatos , Quinases Associadas a rho , Camundongos , Animais , Palmitatos/farmacologia , Palmitatos/metabolismo , Quinases Associadas a rho/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Macrófagos/metabolismo , Inflamação/metabolismo , Obesidade/metabolismo
12.
Molecules ; 27(22)2022 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-36432171

RESUMO

In the presented study, the effects of ROCK inhibitor Y-27632, antifreeze protein III, and boron at two different doses were investigated on the spermatological parameters of Ankara buck semen after freeze-thawing. Ejaculates were collected from bucks using an electroejaculator during the breeding season. The ejaculates that showed appropriate characteristics were pooled and used in the dilution and freezing of semen. The extender groups were formed by adding two different doses of three different additives (ROCK inhibitor Y-27632, 5 and 20 µM; antifreeze protein III, 1 and 4 µg/mL; boron, 0.25 and 1 mM) to the control extender. The semen was diluted with the different extenders at 35-37 °C and loaded into straws. Sperm samples frozen in liquid nitrogen vapors, following equilibration, were stored in liquid nitrogen. It was observed that extender supplementation improved post-thaw motility of Ankara buck semen after freeze-thawing. Differences were significant (p < 0.01) for 5 and 10 µM doses of ROCK inhibitor (71.82% and 74.04 % motility), as well as for 0.25 and 1 mM doses of boron (76.36% and 72.08% motility), compared to the control group (66.15% motility). With respect to the evaluation of acrosomal integrity and mitochondrial activity after freeze-thawing, although supplementation provided protection at all doses, the efficacy was not statistically significant (p > 0.05). It was observed that DNA damage was improved by antifreeze protein III at 1 µg/mL (1.23% ± 0.23%) and by boron at all doses (0.25 mM: 1.83% and 1 mM: 1.18%) compared to the control group (3.37%) (p < 0.01), following the thawing process. In the present study, it was determined that some additives added to the extender provided significant improvements in buck spermatozoa motility and DNA damage after thawing.


Assuntos
Preservação do Sêmen , Sêmen , Masculino , Humanos , Preservação do Sêmen/métodos , Boro/farmacologia , Boro/metabolismo , Quinases Associadas a rho/metabolismo , Criopreservação/métodos , Crioprotetores/farmacologia , Proteínas Anticongelantes/metabolismo , Nitrogênio/metabolismo
13.
Anal Cell Pathol (Amst) ; 2022: 7534181, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36247873

RESUMO

Tetrandrine (Tet), a compound found in a traditional Chinese medicine, presents the protective effect for kidney function. Our study is aimed at clarifying the efficacy and underlying mechanism of Tet on podocyte injury. In this study, podocyte injury was induced in rats with adriamycin (ADR), and MPC5 podocytes were constructed with TRPC6 overexpression. We found that Tet treatment reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen and increased plasma albumin levels in ADR-induced rats. Tet reduced intracellular Ca2+ influx and apoptosis in MPC5 podocytes overexpressing TRPC6. Tet downregulated the expression of renal TRPC6, RhoA, and ROCK1 and upregulated the expression of synaptopodin; meanwhile, it reduced calcineurin activity in vivo and in vitro. In conclusion, Tet protects against podocyte by affecting TRPC6 and its downstream RhoA/ROCK1 signaling pathway.


Assuntos
Podócitos , Animais , Benzilisoquinolinas , Calcineurina/metabolismo , Calcineurina/farmacologia , Creatinina , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Podócitos/metabolismo , Ratos , Albumina Sérica/metabolismo , Albumina Sérica/farmacologia , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/farmacologia , Canal de Cátion TRPC6/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo
14.
Mediators Inflamm ; 2022: 1818758, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36248188

RESUMO

Lysophosphatidic acid (LPA) has disruptive effects on lumbar spinal stenosis (LSS). Recently, LPA has been reported to be involved in spinal cord neuronal injury and toxicity, promoting the pathogenesis of LSS. However, the exact effects of LPA on spinal cord neurons remain unknown. The purpose of this study is to investigate the effects of LPA (18 : 1) on spinal cord neuronal cytotoxicity, apoptosis, DNA damage, and oxidative stress. After clinical detection of LPA secretion, spinal cord neurons were treated with LPA (18 : 1); cell viability was analyzed by MTT assay, and LDH leakage was detected by LDH kit; cell apoptosis was detected by flow cytometry; ROS production was measured by DCFDA staining and MitoSOX Red Staining; the activation of the Gα12/Gα13 signaling pathway was detected by serum response factor response element (SRF-RE) luciferase reporter gene; the relationship among LPA, LPA4/6, and ROCK was examined by western blotting. In spinal cord neurons treated with LPA (18 : 1), cellular activity decreased and LDH release increased. The Rho kinase inhibitor (Y-27632) can attenuate LPA-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons. Moreover mechanistic investigation indicated that LPA (18 : 1) activates Gα12/13-Rho-ROCK2-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons by upregulating LPA4/LPA6 receptors. Further, the Rho kinase inhibitor Y-27632 attenuates the effects of LPA by downregulating LPA4/LPA6 receptors. Taken together, the possible mechanism by which LPA secretion in LSS patients aggravates patient injury was further elucidated using an LPA-induced spinal cord neuronal injury cell model in vitro.


Assuntos
Receptores de Ácidos Lisofosfatídicos , Traumatismos da Medula Espinal , Amidas , Apoptose , Dano ao DNA , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/farmacologia , Humanos , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Neurônios/metabolismo , Estresse Oxidativo , Piridinas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/farmacologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/farmacologia
15.
Sci Rep ; 12(1): 17811, 2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36280692

RESUMO

Rho-associated coiled-coil containing protein kinase 1 (ROCK1) intracellular cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. We observed the activation of ROCK1/myosin light chain (MLC2) signaling pathway in buffalopox virus (BPXV) infected Vero cells. ROCK1 depletion by siRNA and specific small molecule chemical inhibitors (Thiazovivin and Y27632) resulted in a reduced BPXV replication, as evidenced by reductions in viral mRNA/protein synthesis, genome copy numbers and progeny virus particles. Further, we demonstrated that ROCK1 inhibition promotes deadenylation of viral mRNA (mRNA decay), mediated via inhibiting interaction with PABP [(poly(A)-binding protein] and enhancing the expression of CCR4-NOT (a multi-protein complex that plays an important role in deadenylation of mRNA). In addition, ROCK1/MLC2 mediated cell contraction, and perinuclear accumulation of p-MLC2 was shown to positively correlate with viral mRNA/protein synthesis. Finally, it was demonstrated that the long-term sequential passage (P = 50) of BPXV in the presence of Thiazovivin does not select for any drug-resistant virus variants. In conclusion, ROCK1/MLC2 cell signaling pathway facilitates BPXV replication by preventing viral mRNA decay and that the inhibitors targeting this pathway may have novel therapeutic effects against buffalopox.


Assuntos
Vírus Vaccinia , Quinases Associadas a rho , Chlorocebus aethiops , Animais , Quinases Associadas a rho/metabolismo , Vírus Vaccinia/genética , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , RNA Mensageiro/genética , Células Vero , RNA Interferente Pequeno
16.
Oxid Med Cell Longev ; 2022: 7056283, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36275894

RESUMO

Background: A previous study identified miR-451b as a potential biomarker in smoker with or without chronic obstructive pulmonary disease (COPD). However, the function and molecular mechanisms of miR-451b in the pathogenesis of COPD remain elusive. Methods: Macrophages and lung fibroblasts were exposed to 10% cigarette smoke extract (CSE) solution for 24 h. Expression miR-451b and its potential transcription factor p300 were detected. The association between p300 and miR-451b, miR-451b and RhoA was validated by luciferase reporter assay. The release of IL-12 and TNF-αby macrophages was measured by ELISA assay, and Transwell assay was performed to analyze its migration and invasion. Collagen protein of fibroblasts was detected by Western blotting. Results: Results showed that p300 and miR-451b was downregulated, while RhoA was upregulated in CSE-induced macrophages and lung fibroblasts. The stimulation of CSE promoted the degradation of p300 by ubiquitination, and RhoA was confirmed as the target gene of miR-451b. MiR-451b overexpression significantly decreased the release of IL-12 and TNF-α, downregulated the expression of RhoA, ROCK2, and p65, and suppressed cell migration and invasion in CES-induced macrophages. In addition, miR-451b overexpression decreased the expression of RhoA, ROCK2, COL1A1, and COL2A1 in lung fibroblasts. Conclusions: Our data suggest that p300/miR-451b protects against CSE-induced cell stress possibly through downregulating RhoA/ROCK2 pathway.


Assuntos
Fumar Cigarros , MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Humanos , Fator de Necrose Tumoral alfa/metabolismo , Fumar Cigarros/efeitos adversos , Fatores de Transcrição/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Tabaco/efeitos adversos , Biomarcadores , Interleucina-12/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
17.
Int Immunopharmacol ; 113(Pt A): 109199, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36252478

RESUMO

BACKGROUND: Emerging evidence has revealed that circular RNAs (circRNAs) have roles in regulating the complex pathologies of sepsis-induced acute lung injury (sepsis-ALI). Herein, this work aimed to investigate the potential role and mechanism of circPalm2 in the process of sepsis-ALI. METHODS: Primary mice pulmonary microvascular endothelial cells (MPVECs) in functional group were exposed to lipopolysaccharide (LPS). Levels of genes and proteins were measured by qRT-PCR and western blotting. Functional experiments were conducted using In vitro functional experiments were conducted using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry and ELISA analysis. The binding between miR-450b-5p and circPalm2 or ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1) was validated using dual-luciferase reporter, RNA immunoprecipitation and pull-down assays. RESULTS: LPS treatment caused the increase of circPalm2 and ROCK1, as well as the decrease of miR-450b-5p in MPVECs. Knockdown of circPalm2 attenuated LPS-induced proliferation arrest, apoptosis, and production of proinflammatory cytokine IL-6, IL-ß and TNF-α in MPVECs. Mechanistically, circPalm2 sequestered miR-450b-5p to up-regulate ROCK1 expression, revealing the circPalm2/miR-450b-5p/ROCK1 feedback loop. Moreover, the protective functions mediated by circPalm2 silencing on MPVECs under LPS exposure were abolished by miR-129-5p inhibition or ROCK1 overexpression. CONCLUSION: CircPalm2 knockdown can alleviate LPS-evoked MPVEC apoptosis and inflammation via miR-450b-5p/ROCK1 axis, suggesting the potential involvement of this ceRNA network in sepsis-ALI and a broader approach for the therapy of sepsis-ALI.


Assuntos
Lesão Pulmonar Aguda , MicroRNAs , RNA Circular , Sepse , Quinases Associadas a rho , Animais , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Apoptose , Células Endoteliais/metabolismo , Inflamação/genética , Inflamação/complicações , Lipopolissacarídeos , MicroRNAs/genética , MicroRNAs/metabolismo , Sepse/complicações , RNA Circular/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
18.
Sci Rep ; 12(1): 17903, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36284153

RESUMO

Exercise training (ExT) is capable of improving the heart function of spontaneously hypertensive rats (SHRs), but the underlying molecular mechanisms remain elusive. This study was aimed to investigate whether inhibition of RhoA/ROCK signaling pathway contributes to the cardiac protection by low-intensity ExT in SHRs. The results demonstrated that, compared with Wistar-Kyoto (WKY) rats, SHRs obviously exhibited higher blood pressure, increased heart weight index and thickness of left ventricular wall, decreased left ventricular function, damaged myocardial construction, and increased collagen fiber of left ventricle (P < 0.05 or P < 0.01). Meanwhile, the mRNA and protein expression levels of RhoA and ROCK in the heart of SHRs were significantly increased, compared with those of WKY rats (P < 0.05 or P < 0.01). Interestingly, the pathological changes of heart aforementioned were all improved in SHR-ExT rats compared with SHR-Sed rats (P < 0.05 or P < 0.01), indicating the cardiac protection of exercise training. In addition, the cardiac protective effect of exercise training could be blocked by LPA, an activator of Rho/ROCK signaling, and the protective effect in SHR rats could be mimicked by Fasudil, an inhibitor of Rho/ROCK signaling. The results strongly suggest that low-intensity ExT can protect heart against structure and function through inhibiting Rho/ROCK signaling pathway in hypertensive rats.


Assuntos
Hipertensão , Animais , Ratos , Pressão Sanguínea/fisiologia , Colágeno/farmacologia , Hipertensão/patologia , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
19.
Biomolecules ; 12(10)2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36291557

RESUMO

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood-brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB's integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100-300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.


Assuntos
Barreira Hematoencefálica , Glioblastoma , Animais , Camundongos , Ratos , Barreira Hematoencefálica/metabolismo , Quinases Associadas a rho/metabolismo , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Glioblastoma/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico
20.
Cells ; 11(19)2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36231141

RESUMO

Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics. Porcine embryos were generated by parthenogenetic electric activation in feeder-free culture conditions supplemented with or without Endo-EVs. The cellular uptake of Endo-EVs was confirmed using the lipophilic dye PKH26. Endo-EVs cargo contained miR-100, miR-132, and miR-155, together with the mRNAs of porcine endogenous retrovirus (PERV) and ß-catenin. Targeting PERV with CRISPR/Cas9 resulted in reduced expression of PERV mRNA transcripts and increased miR-155 in the Endo-EVs, and supplementing these in embryos reduced embryo attachment. Supplementing the medium containing Endo-EVs with miR-155 inhibitor significantly improved the embryo attachment with a few outgrowths, while supplementing with Rho-kinase inhibitor (RI, Y-27632) dramatically improved both embryo attachment and outgrowths. Moreover, the expression of miR-100, miR-132, and the mRNA transcripts of BCL2, zinc finger E-box-binding homeobox 1, ß-catenin, interferon-γ, protein tyrosine phosphatase non-receptor type 1, PERV, and cyclin-dependent kinase 2 were all increased in embryos supplemented with Endo-EVs + RI compared to those in the control group. Endo-EVs + RI reduced apoptosis and increased the expression of OCT4 and CDX2 and the cell number of embryonic outgrowths. We examined the individual and combined effects of RI compared to those of the miR-155 mimic and found that RI can alleviate the negative effects of the miR-155 mimic on embryo attachment and outgrowths. EVs can improve embryo attachment and the unwanted effects of the de trop cargo contents (miR-155) can be alleviated through anti-apoptotic molecules such as the ROCK inhibitor.


Assuntos
Vesículas Extracelulares , MicroRNAs , Amidas , Animais , Quinase 2 Dependente de Ciclina/metabolismo , Endométrio/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Interferon gama/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piridinas , RNA Mensageiro/metabolismo , Suínos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismo
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