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1.
Chem Biol Interact ; 348: 109625, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34416245

RESUMO

Catalpol is an iridoid glycoside compound isolated from the root of Rehmannia glutinosa, which has been reported to be a promising candidate for the treatment of diabetic diseases. The present study aimed at investigating the effects and potential mechanism of catalpol on endothelial dysfunction and inflammation in diabetic nephropathy (DN). We constructed DN mice and advanced glycation end products (AGEs)-induced mouse glomerular endothelial cells (mGECs) injury model. The results demonstrated that catalpol effectively improved renal pathology and decreased levels of urine protein, serum creatinine, and blood urea nitrogen in DN mice. Catalpol significantly reduced endothelial dysfunction and inflammatory infiltration of macrophages in DN mice and AGEs-induced mGECs. To further study the protective mechanism of catalpol, we transfected DN mice with recombinant adeno-associated virus expressing receptor of AGEs (RAGE) and intervened AGEs-induced mGECs with inhibitors. Catalpol reversed endothelial dysfunction and inflammation aggravated by RAGE overexpression in DN mice. Meanwhile, catalpol significantly inhibited the RAGE/Ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) pathway in DN mice with RAGE overexpression. Moreover, the combination of catalpol with inhibitors of RAGE, RhoA and ROCK exerted stronger anti-endothelial dysfunction and anti-macrophage infiltration effects on AGEs-induced mGECs compared with catalpol alone. In short, this study indicated that catalpol could ameliorate endothelial dysfunction and inflammation via suppression of RAGE/RhoA/ROCK pathway, hereby delaying the progression of DN.


Assuntos
Nefropatias Diabéticas/patologia , Endotélio/efeitos dos fármacos , Glucosídeos Iridoides/farmacologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Animais , Nefropatias Diabéticas/tratamento farmacológico , Endotélio/patologia , Inflamação/complicações , Glucosídeos Iridoides/uso terapêutico , Macrófagos/efeitos dos fármacos , Camundongos , Proteína rhoA de Ligação ao GTP/metabolismo
2.
Biomolecules ; 11(8)2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34439742

RESUMO

Angiotensin II (Ang II) induces vasoconstriction through myosin light chain (MLC) kinase activation and MLC phosphatase inactivation via phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) by Rho kinase. However, the detailed mechanism underlying Rho kinase activation by Ang II is still unknown. We investigated the mechanism of Ang II-induced vasoconstriction mediated by Rho kinase in pressure-overloaded rat thoracic aortas. Pressure-overloaded rats were produced by coarctation of the suprarenal abdominal aorta in four-week-old male Wistar rats. The contractile response to Ang II was significantly enhanced in the pressure-overloaded rats. Ang II-induced vasoconstriction was attenuated by inhibitors of Rho kinase, extracellular signal-regulated kinase 1 and 2 (Erk1/2), and epidermal growth factor receptor (EGFR) in both the sham-operated and pressure-overloaded rats. The Ang II-induced vasoconstriction was attenuated by a Janus kinase 2 (JAK2) inhibitor in only the pressure-overloaded rats. The protein levels of MYPT1 and JAK2 increased only in the pressure-overloaded rat thoracic aortas. These results suggested that Ang II-induced contraction is mediated by Rho kinase activation via EGFR, Erk1/2, and JAK2 in pressure-overloaded rat thoracic aortas. Moreover, Ang II-induced contraction was enhanced in pressure-overloaded rats probably because the protein levels of MYPT1 and JAK2 increased in the thoracic aortas.


Assuntos
Angiotensina II/fisiologia , Aorta Torácica/metabolismo , Vasoconstrição , Quinases Associadas a rho/metabolismo , Animais , Aorta Torácica/patologia , Masculino , Ratos , Ratos Wistar
3.
Arch Biochem Biophys ; 710: 109000, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34343486

RESUMO

Impaired endothelium-mediated vasodilation and/or increased sensitivity to vasoconstrictors lead to vascular smooth muscle cell (VSMC) dysfunction in individuals with diabetes. Diabetic nephropathy is associated with a considerably higher risk of cardiovascular disease and death than their nondiabetic counterparts. We studied the activity of Cullin 3 RING ubiquitin ligase (CRL3) and its substrates in mice using an intraperitoneal injection of streptozotocin (STZ) and db/db mice. The levels of CRL3 adaptors, including Kelch-like 2/3 (KLHL2/3) and Rho-related BTB domain-containing protein 1, were significantly decreased in the aortic tissues and heart of the STZ group, whereas the levels of Cullin 3 (CUL3) and its neddylated derivatives were substantially increased. Decreased KLHL3 expression and significantly increased expression of NEDD8 conjugates were observed in the kidneys of db/db mice. The neddylation inhibitor MLN4924 decreased the degradation of KLHL2/KLHL3 under high-glucose conditions with/without insulin, and transfection with KLHL2 promoted the degradation of its substrates with-no-lysine (WNK) kinases. Increased abundance of WNK3, RhoA/ROCK activity and phosphodiesterase 5 enhanced the sensibility to vasoconstrictors and impaired vasodilation. Moreover, WNK3 localized in VSMCs undergoing cell division, and high-glucose medium increased WNK3 signaling in VSMCs undergoing mitosis, which might explain the increased thickness of aortic tissues in subjects with diabetes. Increases in WNK4 abundance resulted in increased sodium reabsorption in the distal renal tubules. Thus, KLHL2/RhoBTB1/KLHL3 inactivation in the aortic tissues and kidney is a result of excessive activation of neddylation in hyperglycemia and hyperinsulinemia, which affects vascular tone and sodium reabsorption.


Assuntos
Proteínas Culina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Vasoconstrição/fisiologia , Animais , Aorta/metabolismo , Aorta/patologia , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
4.
Biomed Res Int ; 2021: 5561221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34414236

RESUMO

Brucea javanica oil (BJO) is beneficial for the treatment of ulcerative colitis (UC), and that quassinoids in particular brusatol are bioactive components. However, it is still uncertain whether or not other components in BJO, such as oleic acid and fatty acids, have an anti-UC effect. The present study is aimed at comparing the anti-UC effects between brusatol-enriched BJO (BE-BJO) and brusatol-free BJO (BF-BJO) and at exploring the effects and mechanisms of BE-BJO on colon inflammation and intestinal epithelial barrier function. Balb/C mice received 3% (wt/vol) DSS for one week to establish the UC model. Different doses of BE-BJO, BF-BJO, or BJO were treated. The result illustrated that BE-BJO alleviated DSS-induced loss of body weight, an increase of disease activity index (DAI), and a shortening of colon, whereas BF-BJO did not have these protective effects. BE-BJO treatment improved the morphology of colon tissue, inhibited the production and release of TNF-α, IFN-γ, IL-6, and IL-1ß in the colon tissue, and reversed the decreased expressions of ZO-1, occludin, claudin-1, and E-cadherin induced by DSS but augmented claudin-2 expression. Mechanistically, BE-BJO repressed phosphorylation of NF-κB subunit p65, suppressed RhoA activation, downregulated ROCK, and prevented phosphorylation of myosin light chain (MLC) in DSS-treated mice, indicating that the protective effect of BE-BJO is attributed to suppression of NF-κB and RhoA/ROCK signaling pathways. These findings confirm that brusatol is an active component from BJO in the treatment of UC.


Assuntos
Brucea/química , Colite/tratamento farmacológico , Sulfato de Dextrana/efeitos adversos , Óleos Vegetais/administração & dosagem , Quassinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Colite/induzido quimicamente , Colite/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Óleos Vegetais/química , Óleos Vegetais/farmacologia , Quassinas/farmacologia , Resultado do Tratamento , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
5.
Cell Prolif ; 54(9): e13104, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34323331

RESUMO

OBJECTIVES: RAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP-bound inactive state and a GTP-bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation. MATERIALS AND METHODS: Microinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real-time RT-PCR were utilized for the study. RESULTS: Our results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK-cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc-Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection. CONCLUSIONS: Taken together, our results suggest that RAB14 affects ROCK-cofilin pathway for actin-based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.


Assuntos
Meiose/fisiologia , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Actinas , Animais , Citoplasma/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo
6.
Biomed Res Int ; 2021: 5516078, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307654

RESUMO

Background: Despite recent advances in scientific knowledge and clinical practice, management, and treatment of breast cancer, as one of the leading causes of female mortality, breast cancer remains a major burden. Recently, methods employing stem cells and their derivatives, i.e., exosomes, in gene-based therapies hold great promise. Since these natural nanovesicles are able to transmit crucial cellular information which can be engineered to have robust delivery and targeting capacity, they are considered one of the modes of intercellular communication. miR-145, one of the downregulated microRNAs (miRNAs) in various cancers, can regulate tumor cell invasion, metastasis, apoptosis, and proliferation and stem cell differentiation. Objectives: The aim of this study was to investigate the role of exosomes secreted from adipose tissue-derived mesenchymal stem cells (MSCs) for miR-145 transfection into breast cancer cells in order to weaken their expansion and metastasis. Methods: Here, we exploited the exosomes from adipose tissue-derived mesenchymal stem cells (MSC-Exo) to deliver miR-145 in the T-47D breast cancer cell line. Lentiviral vectors of miR-145-pLenti-III-enhanced green fluorescent protein (eGFP) and empty pLenti-III-eGFP as the backbone were used to transfect MSCs and T-47D cells. In order to find the efficiency of exosomes as a delivery vehicle, the expression level of some miR-145 target genes, including Rho-Associated Coiled-Coil Containing Protein Kinase 1 (ROCK1), Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Matrix Metalloproteinase 9 (MMP9), and Tumor Protein p53 (TP53), was compared in all treatment groups (T-47D cells treated by miR-145-transfected MSCs and their derivatives or their backbone) and control group (untransfected T-47D cells) using real-time PCR. Results: The obtained data represented the inhibitory effect of miR-145 on apoptosis induction and metastasis in both direct miR-treated groups. However, exosome-mediated delivery caused an improved anticancer property of miR-145. Conclusion: Restoration of miR-145 using MSC-Exo can be considered a potential novel therapeutic strategy in breast cancer in the future.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Transfecção , Tecido Adiposo/citologia , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Exossomos/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , Metástase Neoplásica , Receptor ErbB-2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
7.
Cell Death Dis ; 12(7): 688, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244467

RESUMO

The insulin-like growth factor 1 receptor (IGF-1R) signaling in cardiomyocytes is implicated in physiological hypertrophy and myocardial aging. Although fibroblasts account for a small amount of the heart, they are activated when the heart is damaged to promote cardiac remodeling. However, the role of IGF-1R signaling in cardiac fibroblasts is still unknown. In this study, we investigated the roles of IGF-1 signaling during agonist-induced cardiac fibrosis and evaluated the molecular mechanisms in cultured cardiac fibroblasts. Using an experimental model of cardiac fibrosis with angiotensin II/phenylephrine (AngII/PE) infusion, we found severe interstitial fibrosis in the AngII/PE infused myofibroblast-specific IGF-1R knockout mice compared to the wild-type mice. In contrast, low-dose IGF-1 infusion markedly attenuated AngII-induced cardiac fibrosis by inhibiting fibroblast proliferation and differentiation. Mechanistically, we demonstrated that IGF-1-attenuated AngII-induced cardiac fibrosis through the Akt pathway and through suppression of rho-associated coiled-coil containing kinases (ROCK)2-mediated α-smooth muscle actin (αSMA) expression. Our study highlights a novel function of the IGF-1/IGF-1R signaling in agonist-induced cardiac fibrosis. We propose that low-dose IGF-1 may be an efficacious therapeutic avenue against cardiac fibrosis.


Assuntos
Actinas/metabolismo , Cardiomiopatias/prevenção & controle , Fibroblastos/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/administração & dosagem , Miócitos Cardíacos/efeitos dos fármacos , Angiotensina II , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Infusões Intravenosas , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenilefrina , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo
8.
Cell Death Dis ; 12(7): 702, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262023

RESUMO

Acquired resistance to glucocorticoids (GCs) is an obstacle to the effective treatment of leukemia, but the molecular mechanisms of steroid insensitivity have not been fully elucidated. In this study, we established an acquired GC-resistant leukemia cell model and found a long noncoding RNA, HOTAIRM1, was overexpressed in the resistant cells by transcriptional profiling, and was higher expressed in patients with poor prognosis. The whole-genome-binding sites of HOTAIRM1 were determined by ChIRP-seq (chromatin isolation by RNA purification combined with sequencing) analysis. Further study determined that HOTAIRM1 bound to the transcriptional inhibitory region of ARHGAP18 and repressed the expression of ARHGAP18, which led to the increase of RHOA/ROCK1 signaling pathway and promoted GC resistance through antiapoptosis of leukemia cells. The inhibition of ROCK1 in GC-resistant cells could restore GCs responsiveness. In addition, HOTAIRM1 could also act as a protein sequester to prevent transcription factor AML1(acute myeloid leukemia 1) from binding to the regulatory region of ARHGAP18 by interacting with AML1. At last, we also proved AML1 could directly activate the expression of HOTAIRM1 through binding to the promoter of HOTAIRM1, which enriched the knowledge on the regulation of lncRNAs. This study revealed epigenetic causes of glucocorticoid resistance from the perspective of lncRNA, and laid a foundation for the optimization of glucocorticoid-based leukemia treatment strategy in clinic.


Assuntos
Antineoplásicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas Ativadoras de GTPase/metabolismo , Glucocorticoides/farmacologia , Leucemia/tratamento farmacológico , MicroRNAs/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Ativadoras de GTPase/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Leucemia/enzimologia , Leucemia/genética , Leucemia/patologia , MicroRNAs/genética , Ligação Proteica , Transdução de Sinais , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genética
9.
JCI Insight ; 6(17)2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314391

RESUMO

Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.


Assuntos
COVID-19/metabolismo , Endotélio Vascular/metabolismo , Ácido Hialurônico/metabolismo , Idoso , COVID-19/sangue , COVID-19/patologia , Citocinas/sangue , Endotélio Vascular/patologia , Feminino , Glicocálix/metabolismo , Glicocálix/patologia , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/sangue , Hialuronoglucosaminidase/sangue , Hialuronoglucosaminidase/metabolismo , Masculino , Pessoa de Meia-Idade , Quinases Associadas a rho/metabolismo
10.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298955

RESUMO

PURPOSE: The effects of Rho-associated coiled-coil containing protein kinase (ROCK) 1 and 2 inhibitor, ripasudil hydrochloride hydrate (Rip), ROCK2 inhibitor, KD025 or rosiglitazone (Rosi) on two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) treated by transforming growth factor (TGFß2) were studied. METHODS: Two-dimension and three-dimension cultured HconF were examined by transendothelial electrical resistance (TEER, 2D), size and stiffness (3D), and the expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, fibronectin (FN), and α-smooth muscle actin (αSMA) by quantitative PCR (2D, 3D) in the presence of Rip, KD025 or Rosi. RESULTS: TGFß2 caused a significant increase in (1) the TEER values (2D) which were greatly reduced by Rosi, (2) the stiffness of the 3D organoids which were substantially reduced by Rip or KD025, and (3) TGFß2 induced a significant up-regulation of all ECMs, except for COL6 (2D) or αSMA (3D), and down-regulation of COL6 (2D). Rosi caused a significant up-regulation of COL1, 4 and 6 (3D), and down-regulation of COL6 (2D) and αSMA (3D). Most of these TGFß2-induced expressions in the 2D and αSMA in the 3D were substantially inhibited by KD025, but COL4 and αSMA in 2D were further enhanced by Rip. CONCLUSION: The findings reported herein indicate that TGFß2 induces an increase in fibrogenetic changes on the plane and in the spatial space, and are inhibited by Rosi and ROCK inhibitors, respectively.


Assuntos
Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Rosiglitazona/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Actinas/biossíntese , Linhagem Celular , Colágeno/biossíntese , Fibronectinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinases Associadas a rho/metabolismo
11.
Brain Behav ; 11(8): e2266, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34156163

RESUMO

AIM: RhoA/Rho kinase pathway is essential for regulating cytoskeletal structure. Although its effect on normal neurite outgrowth has been demonstrated, the role of this pathway in seizure-induced neurite injury has not been revealed. The research examined the phosphorylation level of RhoA/Rho kinase signaling pathway and to clarify the effect of fasudil on RhoA/Rho kinase signaling pathway and neurite outgrowth in kainic acid (KA)-treated Neuro-2A cells and hippocampal neurons. METHOD: Western blotting analysis was used to investigate the expression of key proteins of RhoA/Rho kinase signaling pathway and the depolymerization of actin. After incubated without serum to induce neurite outgrowth, Neuro-2A cells were fixed, and immunofluorescent assay of rhodamine-phalloidin was applied to detect the cellular morphology and neurite length. The influence of KA on neurons was detected in primary hippocampal neurons. Whole-cell patch clamp was conducted in cultured neurons or hippocampal slices to record action potentials. RESULT: KA at the dose of 100-200 µmol/L induced the increase in phosphorylation of Rho-associated coiled-coil-containing protein kinase and decrease in phosphorylation of Lin11, Isl-1 and Mec-3 kinase and cofilin. The effect of 200 µmol/L KA was peaked at 1-2 hours, and then gradually returned to baseline after 8 hours. Pretreatment with Rho kinase inhibitor fasudil reversed KA-induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of slingshot and 14-3-3, which consequently reduced the ratio of G/F-actin. KA treatment induced inhibition of neurite outgrowth and decrease in spines both in Neuro-2a cells and in cultured hippocampal neurons, and pretreatment with fasudil alleviated KA-induced neurite outgrowth inhibition and spine loss. CONCLUSION: These data indicate that inhibiting RhoA/Rho kinase pathway might be a potential treatment for seizure-induced injury.


Assuntos
Neuritos , Quinases Associadas a rho , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Ácido Caínico/toxicidade , Neuritos/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo
12.
Am J Physiol Gastrointest Liver Physiol ; 321(1): G87-G97, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075793

RESUMO

Aging can lead to rectoanal incontinence due to internal anal sphincter (IAS) dysfunction, which is characterized by a decrease in IAS tone and contractility and an increase in nonadrenergic noncholinergic (NANC) relaxation. We aimed to determine whether brain-derived neurotropic factor (BDNF) rescues this aging-associated IAS dysfunction (AAID). To do so, we studied the effects of BDNF on the basal and G protein-coupled receptors (GPCR)-stimulated IAS smooth muscle tone and on NANC relaxation in Fischer 344 rats representing different age groups [26-mo-old (aging) vs. 6-mo-old (young)], before and after tyrosine kinase receptor B (TrkB) antagonist K252a. We also used isolated smooth muscle cells (SMCs) to determine the effects of BDNF before and after different agonists. For some studies, we monitored NO release using smooth muscle perfusates. BDNF reversed AAID by rescuing the basal IAS tone and agonists [thromboxane A2 analog (U46619) and angiotensin II (ANG II)]-induced contractility, and NANC relaxation. These rescue effects of BDNF were selective as K252a attenuated the changes in the IAS without modifying the effects of K+depolarization. Because of the direct association between the basal and GPCR-stimulated IAS tone and RhoA/ROCK activation, we speculate that this pathway in the rescue effects of BDNF. Conversely, our data suggest that aging-associated increased NANC relaxation is reversed by decreased release of NO and decrease in the sensitivity of the released inhibitory neurotransmitter. In summary, BDNF rescue of AAID involves RhoA/ROCK and inhibitory neurotransmission. These data have direct implications for the role of BDNF in the pathophysiology and therapeutic targeting of aging-associated rectoanal motility disorders.NEW & NOTEWORTHY These studies demonstrate that brain-derived neurotropic factor (BDNF) rescues the aging-associated internal anal sphincter (IAS) dysfunction, characterized by a decrease in IAS tone, and increase in non-adrenergic noncholinergic relaxation. We determined the effects of BDNF on the basal and GPCR (TXA2 and ANG II)-stimulated IAS tone, and on NANC relaxation, before and after TrkB inhibitor K252a. BDNF may have an important role in the pathophysiology and therapeutic targeting of certain rectoanal motility disorders.


Assuntos
Envelhecimento/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Masculino , Relaxamento Muscular/efeitos dos fármacos , Tono Muscular/fisiologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo
13.
Biomed Res Int ; 2021: 5565748, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34095301

RESUMO

Gentiopicroside (GPS), a main active secoiridoid glucoside derived from the roots of perennial herbs in the Gentianaceae family, has antispasmodic and relaxant effects. However, the vasorelaxant effects of GPS on aortic rings and the molecular mechanisms involved in these effects are not yet clear. Therefore, we investigated whether GPS inhibits phenylephrine- (PE-) or KCl-induced contractions in isolated rat thoracic aortic rings. The present study found that GPS produced a dose-dependent relaxation in aortic rings precontracted with PE or KCl and significantly reduced CaCl2-, narciclasine- (Rho-kinase activator-), and phorbol-12,13-diacetate- (PKC activator-) induced vasocontractions. Pretreatment with NG-Nitroarginine methyl ester hydrochloride (L-NAME, NOS inhibitor), methylene blue (sGC inhibitor), indomethacin (COX inhibitor), 4-aminopyridine (KV channel inhibitor), and glibenclamide (KATP channel inhibitor) had no influence on the vasorelaxant effect of GPS, while BaCl2 (Kir channel inhibitor), tetraethylammonium chloride (KCa channel inhibitor), ruthenium red (RYR inhibitor), and heparin (IP3R inhibitor) significantly reduced GPS-induced vasorelaxation. Moreover, GPS pretreatment remarkably inhibited the influx of Ca2+ in vascular smooth muscle cells stimulated using KCl or PE-containing CaCl2 solution. Western blot analysis confirmed that GPS treatment inhibited PE-induced increases in the protein levels of p-Akt, p-myosin light chain (MLC), and p-myosin-binding subunit of myosin phosphatase 1 (MYPT1) in the aortic rings. Additionally, the vasorelaxation activity of GPS was attenuated upon pretreatment with LY294002 (PI3K/Akt inhibitor), Y27632 (Rho-kinase inhibitor), and verapamil (L-type Ca2+ channel inhibitor). These findings demonstrate that GPS exhibits endothelium-independent vasorelaxant effects through inhibition of voltage-dependent, receptor-operated, and inositol triphosphate receptor (IP3R)/ryanodine receptor- (RYR-) mediated Ca2+ channels as well as the PI3K/Akt/Rho-kinase signaling pathway.


Assuntos
Aorta Torácica/metabolismo , Glucosídeos Iridoides/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica/efeitos dos fármacos , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , China , Endotélio Vascular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Glucosídeos Iridoides/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Fenilefrina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Cloreto de Potássio/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/fisiologia , Vasodilatação/fisiologia , Vasodilatadores/farmacologia , Quinases Associadas a rho/metabolismo
14.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066490

RESUMO

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. "Adherent" cancer cell lines frequently release "free-floating" cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.


Assuntos
Modelos Biológicos , Neoplasias/patologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metástase Neoplásica , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
15.
EBioMedicine ; 69: 103452, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34186485

RESUMO

BACKGROUND: Intestinal obstruction caused by intestinal fibrosis is a common and serious complication of Crohn's disease (CD). Intestinal fibroblasts, the main effector cells mediating gastrointestinal fibrosis, are activated during chronic inflammation. However, the mechanism of fibroblast activation in CD has not been well elucidated. METHODS: Fibroblasts isolated from stenotic and nonstenotic intestines of CD patients were used for RNA sequencing. Immunohistochemical and immunofluorescent staining was performed to evaluate the correlation between intestinal fibrosis and YAP/TAZ expression in our CD cohort and a DSS-induced chronic colitis murine model. A Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) inhibitor was used to explore the ROCK1-YAP/TAZ axis in intestinal fibroblasts in vitro and DSS-induced chronic colitis murine model in vivo. FINDINGS: The expression of YAP/TAZ was significantly upregulated in stenotic fibroblasts, which was associated with the YAP/TAZ target gene signature. YAP/TAZ knockdown suppressed the activation of intestinal fibroblasts. In intestinal fibroblasts, YAP/TAZ were activated by the Rho-ROCK1 signalling pathway. High YAP/TAZ expression was positively correlated with ROCK1 expression, which is a prognostic marker for intestinal obstruction in CD patients. INTERPRETATION: YAP/TAZ activation can lead to fibroblast activation and intestinal obstruction in CD. The effect of ROCK1 inhibitor on alleviating intestinal fibrosis is associated with YAP/TAZ inhibition. Targeted inhibition of YAP/TAZ in fibroblasts may be a potential therapeutic strategy to suppress intestinal fibrosis in CD. FUNDING: This work was supported by the National Key R&D Program of China (2019YFC1316002), the NSFC (81873547, 82073201, 81874177, 82000481) and the Shanghai Sailing Program (20YF1429400).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Crohn/metabolismo , Fibroblastos/metabolismo , Obstrução Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Animais , Células Cultivadas , Doença de Crohn/complicações , Doença de Crohn/patologia , Feminino , Fibrose , Humanos , Obstrução Intestinal/etiologia , Obstrução Intestinal/patologia , Intestinos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fatores de Transcrição/genética , Quinases Associadas a rho/metabolismo
16.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063504

RESUMO

Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in various cell models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms regulating oligodendrocyte differentiation after PKC activation are still unclear. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to provide a comprehensive analysis of the proteome expression changes in the MO3.13 oligodendroglial cell line after PKC activation. Our findings suggest that multiple networks that communicate and coordinate with each other may finally determine the fate of MO3.13 cells, thus identifying a modular and functional biological structure. In this work, we provide a detailed description of these networks and their participating components and interactions. Such assembly allows perturbing each module, thus describing its physiological significance in the differentiation program. We applied this approach by targeting the Rho-associated protein kinase (ROCK) in PKC-activated cells. Overall, our findings provide a resource for elucidating the PKC-mediated network modules that contribute to a more robust knowledge of the molecular dynamics leading to this cell fate transition.


Assuntos
Diferenciação Celular/fisiologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Proteína Quinase C/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Espectrometria de Massas/métodos , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Quinases Associadas a rho/metabolismo
17.
Neurochem Res ; 46(8): 2167-2180, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34037903

RESUMO

Long non-coding RNAs (lncRNAs) are considered as critical regulators in the pathogenesis of cerebral ischemia. In this present study, we aimed to investigate the impact and underlying mechanism of lncRNA X-inactive specific transcript (XIST) in cerebral ischemia/reperfusion (I/R) injury. An oxygen-glucose deprivation/reperfusion (OGD/R) model in PC12 cells was applied to mimic cerebral I/R injury in vitro and middle cerebral artery occlusion/reperfusion (MCAO/R) model was performed in mice to mimic cerebral I/R injury in vivo. Real-time PCR, fluorescence in situ hybridization (FISH) assay, and western blotting assay were carried out to detect the expression levels of XIST, miR-362, and Rho-related coiled-coil containing protein kinase 2 (ROCK2). The functional experiments were measured by CCK-8 assay, immumofluorescence assay, ELISA assay, TUNEL, and TTC staining. Results displayed that XIST was elevated in PC12 cells with OGD/R, as well as in the ischemic penumbra of mice with MCAO/R. In vitro, knockdown of XIST facilitated cell survival, inhibited apoptosis, and alleviated inflammation injury in OGDR PC12 cells. In vivo, inhibition of XIST remarkably reduced the neurological impairments, promoted neuron proliferation, and suppressed apoptosis in MCAO mice. Mechanistically, XIST acted as a competing endogenous RNA of miR-362 to regulate the downstream gene ROCK2. In conclusion, depletion of XIST attenuated I/R-induced neurological impairment and inflammatory response via the miR-362/ROCK2 axis. These findings offer a potential novel strategy for ischemic stroke therapy.


Assuntos
Infarto da Artéria Cerebral Média/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Hipóxia Celular/fisiologia , Técnicas de Silenciamento de Genes , Glucose/deficiência , Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Células PC12 , RNA Longo não Codificante/genética , Ratos
18.
Nat Methods ; 18(5): 528-541, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33941937

RESUMO

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Polifarmacologia , Técnicas de Cultura de Células , Criopreservação/métodos , Crioprotetores/química , Regulação da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
19.
Aging (Albany NY) ; 13(11): 14940-14967, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031265

RESUMO

Dysregulation of transcriptome expression has been reported to play an increasingly significant role in AD. In this study, we firstly identified a vital gene module associated with the accumulation of ß-amyloid (Aß) and phosphorylated tau (p-tau) using the WGCNA method. The vital module, named target module, was then employed for the identification of key transcriptome biomarkers. For coding RNA, GNA13 and GJA1 were identified as key biomarkers based on ROC analysis. As for non-coding RNA, MEG3, miR-106a-3p, and miR-24-3p were determined as key biomarkers based on analysis of a ceRNA network and ROC analysis. Experimental analyses firstly confirmed that GNA13, GJA1, and ROCK2, a downstream effector of GNA13, were all increased in 5XFAD mice, compared to littermate mice. Moreover, their expression was increased with aging in 5XFAD mice, as Aß and p-tau pathology developed. Besides, the expression of key ncRNA biomarkers was verified to be decreased in 5XFAD mice. GSEA results indicated that GNA13 and GJA1 were respectively involved in ribosome and spliceosome dysfunction. MEG3, miR-106a-3p, and miR-24-3p were identified to be involved in MAPK pathway and PI3K-Akt pathway based on enrichment analysis. In summary, we identified several key transcriptome biomarkers, which promoted the prediction and diagnosis of AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Redes Reguladoras de Genes , Transcriptoma/genética , Animais , Cromossomos de Mamíferos/genética , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
20.
Chem Biol Interact ; 344: 109529, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34029542

RESUMO

Ganoderic acid A (GAA), one of the major triterpenoid components extracted from Ganoderma mushroom has been shown to possess numerous important pharmacological activities. The present study was aimed to investigate the mechanisms of GAA on carbon tetrachloride (CCl4)-induced kidney inflammation, fibrosis and oxidative stress in mice. The male mice were treated with 25 and 50 mg/mg GAA after stimulated with CCl4. Our results showed that GAA improved renal damage by decreasing the serum levels of creatinine, urea, uric acid and alleviating kidney fibrosis. GAA ameliorated CCl4-induced indices of inflammation. GAA suppressed oxidative stress by regulating the glutathione antioxidant system and the thioredoxin antioxidant system. GAA increased the activations of thioredoxin reductase (TrxR), Trx, GSH, SOD, GPx. Furthermore, GAA supplementation inhibited the JAK and STAT3 pathway. GAA inhibited the activations of RhoA, ROCK, NF-κB, TGF-ß and Smad3. Thus, this study demonstrated that GAA possesses immune-protective properties through regulating the Trx/TrxR, JAK2/STAT3 and RhoA/ROCK pathways.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Fibrose/tratamento farmacológico , Ácidos Heptanoicos/uso terapêutico , Nefropatias/tratamento farmacológico , Lanosterol/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Animais , Tetracloreto de Carbono , Fibrose/induzido quimicamente , Fibrose/patologia , Janus Quinase 2/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/patologia , Lanosterol/uso terapêutico , Masculino , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Proteína Smad3/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Quinases Associadas a rho/metabolismo
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