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The biological role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (Apex1) in modulating systemic inflammation remains unclear. This study aimed to assess the impact of Apex1 deficiency on systemic inflammation triggered by lipopolysaccharide (LPS) in a murine model. The methods involved transcriptomic analysis and assessments of inflammatory responses in age-matched 8-week-old Apex1+/- and wild-type Apex1+/+ mice, generated using the CRISPR/Cas9 system. Apex1+/- mice displayed no overt changes in body weight, however, Apex1 protein expressions in tissues were significantly reduced compared to wild-type mice. Furthermore, in Apex1+/- mice transcriptomic analysis showed that genes associated with antioxidant pathways were downregulated, and levels of superoxide production, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and malondialdehyde (MDA) were increased. Moreover, hematological analysis showed increased neutrophil levels and a twofold increase in the count of splenic lymphocyte antigen 6 family member G+ (Ly6G+) neutrophils in the Apex1+/- mice compared to those in Apex1+/+ mice. Furthermore, following LPS treatment, the levels of cytokines and chemokines, including interleukin-1ß, interleukin-10, tumor necrosis factor-α, and monocyte chemoattractant protein 1, increased in the Apex1+/- mice. The Kaplan-Meier curve showed a significant reduction in the survival rates of Apex1+/- mice treated with LPS compared to those of Apex1+/+ mice. The hepatic and lung injury scores and Ly6G+ neutrophil infiltration levels also increased in Apex1+/- mice after LPS treatment. These results showed that Apex1 deficiency exacerbated the LPS-induced tissue damage in the lung and liver. These findings illustrate that in vivo Apex1 deficiency exacerbates LPS-induced systemic inflammation, tissue damage, and mortality in a murine model, highlighting the crucial role of Apex1 in mitigating inflammatory responses and maintaining a holistic physiological equilibrium.
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BACKGROUND: TRAIL is an anticancer drug that induces cancer cell apoptosis by interacting with death receptors (DRs). However, owing to low cell-surface expression of DRs, certain colorectal cancer (CRC) cells resist TRAIL-induced apoptosis. Newcastle disease virus (NDV) infection can elevate DR protein expression in cancer cells, potentially influencing their TRAIL sensitivity. However, the precise mechanism by which NDV infection modulates DR expression and impacts TRAIL sensitivity in cancer cells remains unknown. METHODS: Herein, we developed nonpathogenic NDV VG/GA strain-based recombinant NDV (rNDV) and TRAIL gene-containing rNDV (rNDV-TRAIL). We observed that viral infections lead to increased DR and TRAIL expressions and activate signaling proteins involved in intrinsic and extrinsic apoptosis pathways. Experiments were conducted in vitro using TRAIL-resistant CRC cells (HT-29) and nonresistant CRC cells (HCT116) and in vivo using relevant mouse models. RESULTS: rNDV-TRAIL was found to exhibit better apoptotic efficacy than rNDV in CRC cells. Notably, rNDV-TRAIL had the stronger cancer cell-killing effect in TRAIL-resistant CRC cells. Western blot analyses showed that both rNDV and rNDV-TRAIL infections activate signaling proteins involved in the intrinsic and extrinsic apoptotic pathways. Notably, rNDV-TRAIL promotes concurrent intrinsic and extrinsic signal transduction in both HCT-116 and HT-29 cells. CONCLUSIONS: Therefore, rNDV-TRAIL infection effectively enhances DR expression in DR-depressed HT-29 cells. Moreover, the TRAIL protein expressed by rNDV-TRAIL effectively interacts with DR, leading to enhanced apoptosis in TRAIL-resistant HT-29 cells. Therefore, rNDV-TRAIL has potential as a promising therapeutic approach for treating TRAIL-resistant cancers.
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Antineoplásicos , Neoplasias Colorretais , Humanos , Animais , Camundongos , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Células HT29 , Células HCT116 , Antineoplásicos/metabolismo , Apoptose , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
NR2D subunit-containing NMDA receptors (NMDARs) gradually disappear during brain maturation but can be recruited by pathophysiological stimuli in the adult brain. Here, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication recruited NR2D subunit-containing NMDARs that generated an Mg2+-resistant tonic NMDA current (INMDA) in dopaminergic (DA) neurons in the midbrain of mature male mice. MPTP selectively generated an Mg2+-resistant tonic INMDA in DA neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Consistently, MPTP increased NR2D but not NR2B expression in the midbrain regions. Pharmacological or genetic NR2D interventions abolished the generation of Mg2+-resistant tonic INMDA in SNpc DA neurons, and thus attenuated subsequent DA neuronal loss and gait deficits in MPTP-treated mice. These results show that extrasynaptic NR2D recruitment generates Mg2+-resistant tonic INMDA and exacerbates DA neuronal loss, thus contributing to MPTP-induced Parkinsonism. The state-dependent NR2D recruitment could be a novel therapeutic target for mitigating cell type-specific neuronal death in neurodegenerative diseases.SIGNIFICANCE STATEMENT NR2D subunit-containing NMDA receptors (NMDARs) are widely expressed in the brain during late embryonic and early postnatal development, and then downregulated during brain maturation and preserved at low levels in a few regions of the adult brain. Certain stimuli can recruit NR2D subunits to generate tonic persistent NMDAR currents in nondepolarized neurons in the mature brain. Our results show that MPTP intoxication recruits NR2D subunits in midbrain dopaminergic (DA) neurons, which leads to tonic NMDAR current-promoting dopaminergic neuronal death and consequent abnormal gait behavior in the MPTP mouse model of Parkinson's disease (PD). This is the first study to indicate that extrasynaptic NR2D recruitment could be a target for preventing neuronal death in neurodegenerative diseases.
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Doença de Parkinson , Receptores de N-Metil-D-Aspartato , Camundongos , Animais , Masculino , Receptores de N-Metil-D-Aspartato/metabolismo , N-Metilaspartato/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , Camundongos Endogâmicos C57BL , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Substância Negra/metabolismoRESUMO
Mitochondrial oxidative phosphorylation (OXPHOS) system dysfunction in cancer cells has been exploited as a target for anti-cancer therapeutic intervention. The downregulation of CR6-interacting factor 1 (CRIF1), an essential mito-ribosomal factor, can impair mitochondrial function in various cell types. In this study, we investigated whether CRIF1 deficiency induced by siRNA and siRNA nanoparticles could suppress MCF-7 breast cancer growth and tumor development, respectively. Our results showed that CRIF1 silencing decreased the assembly of mitochondrial OXPHOS complexes I and II, which induced mitochondrial dysfunction, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential depolarization, and excessive mitochondrial fission. CRIF1 inhibition reduced p53-induced glycolysis and apoptosis regulator (TIGAR) expression, as well as NADPH synthesis, leading to additional increases in ROS production. The downregulation of CRIF1 suppressed cell proliferation and inhibited cell migration through the induction of G0/G1 phase cell cycle arrest in MCF-7 breast cancer cells. Similarly, the intratumoral injection of CRIF1 siRNA-encapsulated PLGA nanoparticles inhibited tumor growth, downregulated the assembly of mitochondrial OXPHOS complexes I and II, and induced the expression of cell cycle protein markers (p53, p21, and p16) in MCF-7 xenograft mice. Thus, the inhibition of mitochondrial OXPHOS protein synthesis through CRIF1 deletion destroyed mitochondrial function, leading to elevated ROS levels and inducing antitumor effects in MCF-7 cells.
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Neoplasias da Mama , Animais , Feminino , Humanos , Camundongos , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/metabolismo , Células MCF-7 , Monoéster Fosfórico Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53 , Polietilenoglicóis/química , NanopartículasRESUMO
Endothelial senescence impairs vascular function and thus is a primary event of age-related vasculature diseases. Isocitrate dehydrogenase 2 (IDH2) plays an important role in inducing alpha-ketoglutarate (α-KG) production and preserving mitochondrial function. However, the mechanism and regulation of IDH2 in endothelial senescence have not been elucidated. We demonstrated that downregulation of IDH2 induced accumulation of miR-34b/c, which impaired mitophagy and elevated mitochondrial reactive oxygen species (ROS) levels by inhibiting mitophagy-related markers (PTEN-induced putative kinase 1 (PINK1), Parkin, LC-II/LC3-I, and p62) and attenuating Sirtuin deacetylation 3 (Sirt3) expression. The mitochondrial dysfunction induced by IDH2 deficiency disrupted cell homeostasis and the cell cycle and led to endothelial senescence. However, miR-34b/c inhibition or α-KG supplementation restored Sirt3, PINK1, Parkin, LC-II/LC3-I, p62, and mitochondrial ROS levels, subsequently alleviating endothelial senescence. We showed that IDH2 played a crucial role in regulating endothelial senescence via induction of miR-34b/c in endothelial cells.
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BACKGROUND: The relationship between autophagy and diabetic peripheral neuropathy (DPN) has been highlighted in few reports. Using an animal model, the authors investigated the relationship between autophagy and DPN, focused particularly on changes in autophagy in Schwann cells. METHODS: The ultrastructural features of DPN mice were evaluated in vivo using transmission electron microscopy. Dysfunction of autophagy in DPN was evaluated using immunofluorescence microscopy and Western blot analysis of proteins related to autophagy, including Beclin1, LC3, and p62. Reactive oxygen species levels were measured in vitro in glucose-treated Schwann cells. Dysfunction of autophagy in glucose-treated Schwann cells was examined by immunofluorescence microscopy and Western blot analysis. RESULTS: Reduced myelin thickness and axonal shrinkage were observed in the sciatic nerves of DPN mice. Reactive oxygen species levels were increased in Schwann cells treated with high glucose ( P < 0.05). The expression of Beclin1 was increased in DPN mice and Schwann cells treated with high glucose ( P < 0.05), whereas the expression of LC3-II/LC3-I ratio and p62 were decreased in DPN mice and Schwann cells treated with high glucose ( P < 0.05). CONCLUSIONS: These results suggest that increased levels of reactive oxygen species induced by high glucose may contribute to autophagy dysfunction in Schwann cells. Autophagy dysfunction especially in Schwann cells may be an underlying cause of DPN. CLINICAL RELEVANCE STATEMENT: This study presents the pathological mechanism of diabetic peripheral neuropathy.
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Diabetes Mellitus , Neuropatias Diabéticas , Camundongos , Animais , Neuropatias Diabéticas/etiologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Beclina-1/metabolismo , Células de Schwann/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Glucose/uso terapêutico , Autofagia/fisiologiaRESUMO
PURPOSE: The isocitrate dehydrogenase (IDH) family plays an essential role in metabolism and energy production. The relative expression levels of IDH isoforms (IDH1, IDH2, and IDH3) have prognostic significance in several malignancies, including breast carcinoma. However, the IDH isozyme expression levels in different cancer stages and types have not been determined in breast carcinoma tissues. METHODS: We analyzed the messenger RNA (mRNA) and protein levels of IDH (IDH1, IDH2, and IDH3A) and α-ketoglutarate (α-KG) in 59 breast carcinoma tissues. RESULTS: The mRNA level of IDH2 was significantly increased at stages 2 and 3 in triple-negative and (ER-/PR-/HER+) breast cancers. However, the elevated α-KG level was only observed in stages 2 and 3, with no differences in the various breast carcinoma types. Western blotting analysis showed that IDH2 protein expression increased in the patient tissues and cell lines. An in vitro study showed IDH2 downregulation in the triple-negative breast cancer cell line MDA-MB-231 that inhibited cell proliferation and migration and induced cell cycle arrest in the G0/G1 phase. CONCLUSION: These findings suggest that different from IDH1 and IDH3, IDH2 is more highly expressed in stages 2 and 3 breast cancer tissues, especially in triple-negative breast cancer. IDH2 potentially serves as a target to detect unknown mechanisms in breast cancer.
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Breast cancer is the most common cancer and the leading cause of cancer-related mortality among females worldwide. Triple-negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and is usually more aggressive and has a poorer prognosis. Sericite has been known to have antitumor and immune-stimulatory effects. Although the chemopreventive potential of sericite has been demonstrated in other cancers, its molecular pathways in TNBC still require investigation. Thus, in the present study, the antitumor mechanism of sericite against MDA-MB231 breast cancer cells was examined in vitro and in an in vivo xenograft mouse model. Sericite treatment reduced cell proliferation and cell proliferation marker proliferating cell nuclear antigen (PCNA) in MDA-MB231 cells. It also decreased the total cell number and arrested cells in the G0/G1 phase of the cell cycle with an increase in the phosphorylation of P53 and upregulation of cell cycle regulatory proteins P21 and P16. In addition, sericite treatment also induced apoptosis signaling, which was evident by the upregulation of apoptotic protein markers cleaved caspases 3 and 9. A reduction in reactive oxygen species (ROS), NADPH oxidase 4 (NOX4), p22phox, and heat shock proteins (HSPs) was also observed. Similar results were obtained in vivo with significantly reduced tumor volume in sericite-administered mice. Collectively, these findings suggest that sericite has antitumor potential based on its property to induce cell cycle arrest and apoptotic cell death and therefore could serve as a potential therapeutic agent and crucial candidate in anticancer drug development for TNBC.
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Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic metabolism dysfunction. However, the mechanistic role of miR204 in the development of NAFLD is unknown. We investigate the functional significance of miR204 in the evolution of NAFLD. IDH2 KO mice feed a normal diet (ND) or HFD increased body weight, epididymal fat-pad weight, lipid droplet in liver, blood parameter and inflammation compared to WT mice fed a ND or HFD. Moreover, the expression of miR204 is increased in mice with IDH2 deficiency. Increased miR204 by IDH2 deficiency regulates carnitine palmitoyltransferase 1a (cpt1a) synthesis, which inhibits fatty acid ß-oxidation. Inhibition of miR204 prevents the disassembly of two fatty acid-related genes by activating CPT1a expression, which decreases lipid droplet in liver, inflammatory cytokines, epididymal fat pad weight, blood parameters. Increased miR204 by IDH2 deficiency promotes the pathogenesis of HFD-induced NAFLD by regulating hepatic fatty acid metabolism and inflammation.
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Hepatopatia Gordurosa não Alcoólica , Animais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Citocinas/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Syndecan-2 (SDC2), a cell-surface heparin sulfate proteoglycan of the glycocalyx, is mainly expressed in endothelial cells. Although oxidative stress and inflammatory mediators have been shown to mediate dysfunction of the glycocalyx, little is known about their role in vascular endothelial cells. In this study, we aimed to identify the mechanism that regulates SDC2 expression in isocitrate dehydrogenase 2 (IDH2)-deficient endothelial cells, and to investigate the effect of ulinastatin (UTI) on this mechanism. We showed that knockdown of IDH2 induced SDC2 expression in human umbilical vein endothelial cells (HUVECs). Matrix metalloproteinase 7 (MMP7) influences SDC2 expression. When IDH2 was downregulated, MMP7 expression was increased, as was TGF-ß signaling, which regulates MMP7. Inhibition of MMP7 activity using MMP inhibitor II significantly reduced SDC2, suggesting that IDH2 mediated SDC2 expression via MMP7. Moreover, expression of SDC2 and MMP7, as well as TGF-ß signaling, increased in response to IDH2 deficiency, and treatment with UTI reversed this increase. Similarly, the increase in SDC2, MMP7, and TGF-ß signaling in the aorta of IDH2 knockout mice was reversed by UTI treatment. These findings suggest that IDH2 deficiency induces SDC2 expression via TGF-ß and MMP7 signaling in endothelial cells.
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Elevated plasma homocysteine levels can induce vascular endothelial dysfunction; however, the mechanisms regulating homocysteine metabolism in impaired endothelial cells are currently unclear. In this study, we deleted the essential mitoribosomal gene CR6 interacting factor 1 (CRIF1) in human umbilical vein endothelial cells (HUVECs) and mice to induce endothelial cell dysfunction; then, we monitored homocysteine accumulation. We found that CRIF1 downregulation caused significant increases in intracellular and plasma concentrations of homocysteine, which were associated with decreased levels of folate cycle intermediates such as 5-methyltetrahydrofolate (MTHF) and tetrahydrofolate (THF). Moreover, dihydrofolate reductase (DHFR), a key enzyme in folate-mediated metabolism, exhibited impaired activity and decreased protein expression in CRIF1 knockdown endothelial cells. Supplementation with folic acid did not restore DHFR expression levels or MTHF and homocysteine concentrations in endothelial cells with a CRIF1 deletion or DHFR knockdown. However, the overexpression of DHFR in CRIF1 knockdown endothelial cells resulted in decreased accumulation of homocysteine. Taken together, our findings suggest that CRIF1-deleted endothelial cells accumulated more homocysteine, compared with control cells; this was primarily mediated by the disruption of DHFR expression.
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Keloids are a common form of pathologic wound healing and are characterized by an excessive production of extracellular matrix. This study examined the major contributing mechanism of human keloid pathogenesis using transcriptomic analysis. We identified the upregulation of mitochondrial oxidative stress response, protein processing in the endoplasmic reticulum, and TGF-ß signaling in human keloid tissue samples compared to controls, based on ingenuity pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Electron microscopic examinations revealed an increased number of dysmorphic mitochondria and expanded endoplasmic reticulum (ER) in human keloid tissue samples than that in controls. Western blot analysis performed using human tissues suggested noticeably higher ER stress signaling in keloids than in normal tissues. Treatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, significantly decreased scar formation in rabbit models, compared to normal saline and steroid injections. In summary, our findings demonstrate the contributions of mitochondrial dysfunction and dysregulated ER stress signaling in human keloid formation and the potential of TUDCA in the treatment of keloids.
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Colagogos e Coleréticos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Queloide/prevenção & controle , Ácido Tauroquenodesoxicólico/farmacologia , Adulto , Animais , Apoptose , Estudos de Casos e Controles , Feminino , Humanos , Queloide/etiologia , Queloide/metabolismo , Queloide/patologia , Masculino , Coelhos , Transdução de SinaisRESUMO
Rho GDP-dissociation inhibitor (RhoGDI), a downregulator of Rho family GTPases, prevents nucleotide exchange and membrane association. It is responsible for the activation of Rho GTPases, which regulate a variety of cellular processes, such as migration. Although RhoGDI2 has been identified as a tumor suppressor gene involved in cellular migration and invasion, little is known about its role in vascular endothelial cell (EC) migration. CR6-interacting factor 1 (CRIF1) is a CR6/GADD45-interacting protein with important mitochondrial functions and regulation of cell growth. We examined the expression of RhoGDI2 in CRIF1-deficient human umbilical vein endothelial cells (HUVECs) and its role in cell migration. Expression of RhoGDI2 was found to be considerably higher in CRIF1-deficient HUVECs along with suppression of cell migration. Moreover, the phosphorylation levels of Akt and CREB were decreased in CRIF1-silenced cells. The Akt-CREB signaling pathway was implicated in the changes in endothelial cell migration caused by CRIF1 downregulation. In addition to RhoGDI2, we identified another factor that promotes migration and invasion of ECs. Adrenomedullin2 (ADM2) is an autocrine/paracrine factor that regulates vascular tone and other vascular functions. Endogenous ADM2 levels were elevated in CRIF1-silenced HUVECs with no effect on cell migration. However, siRNA-mediated depletion of RhoGDI2 or exogenous ADM2 administration significantly restored cell migration via the Akt-CREB signaling pathway. In conclusion, RhoGDI2 and ADM2 play important roles in the migration of CRIF1-deficient endothelial cells.
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Proteínas de Ciclo Celular/genética , Células Endoteliais/citologia , Hormônios Peptídicos/genética , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/deficiência , Movimento Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genéticaRESUMO
Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that can be secreted, and recently suggested as new biomarker for vascular inflammation. However, the endogenous hormones for APE1/Ref-1 secretion and its underlying mechanisms are not defined. Here, the effect of twelve endogenous hormones on APE1/Ref-1 secretion was screened in cultured vascular endothelial cells. The endogenous hormones that significantly increased APE1/Ref-1 secretion was 17ß-estradiol (E2), 5?-dihydrotestosterone, progesterone, insulin, and insulin-like growth factor. The most potent hormone inducing APE1/Ref-1 secretion was E2, which in cultured endothelial cells, E2 for 24 h increased APE1/Ref-1 secretion level of 4.56 ± 1.16 ng/mL, compared to a basal secretion level of 0.09 ± 0.02 ng/mL. Among the estrogens, only E2 increased APE1/Ref-1 secretion, not estrone and estriol. Blood APE1/Ref-1 concentrations decreased in ovariectomized (OVX) mice but were significantly increased by the replacement of E2 (0.39 ± 0.09 ng/mL for OVX vs. 4.67 ± 0.53 ng/mL for OVX + E2). E2-induced APE1/Ref-1secretion was remarkably suppressed by the estrogen receptor (ER) blocker fulvestrant and intracellular Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), suggesting E2-induced APE1/Ref-1 secretion was dependent on ER and intracellular calcium. E2-induced APE1/Ref-1 secretion was significantly inhibited by exosome inhibitor GW4869. Furthermore, APE1/Ref-1 level in CD63-positive exosome were increased by E2. Finally, fluorescence imaging data showed that APE1/Ref-1 co-localized with CD63-labled exosome in the cytoplasm of cells upon E2 treatment. Taken together, E2 was the most potent hormone for APE1/Ref-1 secretion, which appeared to occur through exosomes that were dependent on ER and intracellular Ca2+. Furthermore, hormonal effects should be considered when analyzing biomarkers for vascular inflammation.
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Keloids are a type of aberrant skin scarring characterized by excessive accumulation of collagen and extracellular matrix (ECM), arising from uncontrolled wound healing responses. While typically non-pathogenic, keloids are occasionally regarded as a form of benign tumor. CR6-interacting factor 1 (CRIF1) is a well-known CR6/GADD45-interacting protein, that has both nuclear and mitochondrial functions, and also exerts regulatory effects on cell growth and apoptosis. In this study, cell proliferation, cell migration, collagen production and TGF-ß signaling was compared between normal fibroblasts (NFs) and keloid fibroblasts (KFs). Subsequently, the effects of CRIF1 deficiency were investigated in both NFs and KFs. Cell proliferation, cell migration, collagen production and protein expressions of TGF-ß, phosphorylation of Smad2 and Smad3 were all found to be higher in KFs compared to NFs. CRIF1 deficiency in NFs and KFs inhibited cell proliferation, migration, and collagen production. In addition, phosphorylation of Smad2 and Smad3, which are transcription factors of collagen, was decreased. In contrast, mRNA expression levels of Smad7 and SMURF2, two important inhibitory proteins of Smad2/3, were increased, suggesting that CRIF1 may regulate collagen production. CRIF1 deficiency decreases the proliferation and migration of KFs, thereby inhibiting their overgrowth via the transforming growth factor-ß (TGF-ß)/Smad pathway. CRIF1 may therefore represent a potential therapeutic target in keloid pathogenesis.
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Proteínas de Ciclo Celular/metabolismo , Fibroblastos/patologia , Queloide/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Ciclo Celular , Proteínas de Ciclo Celular/genética , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Queloide/genética , Queloide/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genética , Adulto JovemRESUMO
The CR6-interacting factor1 (CRIF1) mitochondrial protein is indispensable for peptide synthesis and oxidative phosphorylation. Cardiomyocyte-specific deletion of CRIF1 showed impaired mitochondrial function and cardiomyopathy. We developed an endothelial cell-specific CRIF1 deletion mouse to ascertain whether dysfunctional endothelial CRIF1 influences cardiac function and is mediated by the antioxidant protein sirtuin 1 (SIRT1). We also examined the effect of the potent SIRT1 activator SRT1720 on cardiac dysfunction. Mice with endothelial cell-specific CRIF1 deletion showed an increased heart-to-body weight ratio, increased lethality, and markedly reduced fractional shortening of the left ventricle, resulting in severe cardiac dysfunction. Moreover, endothelial cell-specific CRIF1 deletion resulted in mitochondrial dysfunction, reduced ATP levels, inflammation, and excessive oxidative stress in heart tissues, associated with decreased SIRT1 expression. Intraperitoneal injection of SRT1720 ameliorated cardiac dysfunction by activating endothelial nitric oxide synthase, reducing oxidative stress, and inhibiting inflammation. Furthermore, the decreased endothelial junction-associated protein zonula occludens-1 in CRIF1-deleted mice was significantly recovered after SRT1720 treatment. Our results suggest that endothelial CRIF1 plays an important role in maintaining cardiac function, and that SIRT1 induction could be a therapeutic strategy for endothelial dysfunction-induced cardiac dysfunction.
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Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3ß-NF-κB signaling pathway. Ref1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.
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Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is involved in DNA base repair and reducing activity. However, the role of APE1/Ref-1 in atherosclerosis is unclear. Herein, we investigated the role of APE1/Ref-1 in atherosclerotic apolipoprotein E (ApoE-/-) mice fed with a Western-type diet. We found that serologic APE1/Ref-1 was strongly correlated with vascular inflammation in these mice. Neutrophil/lymphocyte ratio (NLR), endothelial cell/macrophage activation, and atherosclerotic plaque formation, reflected by atherosclerotic inflammation, were increased in the ApoE-/- mice fed with a Western-type diet. APE1/Ref-1 expression was upregulated in aortic tissues of these mice, and was co-localized with cells positive for cluster of differentiation 31 (CD31) and galectin-3, suggesting endothelial cell/macrophage expression of APE1/Ref-1. Interestingly, APE1/Ref-1 plasma levels of ApoE-/- mice fed with a Western-type diet were significantly increased compared with those of the mice fed with normal diet (15.76 ± 3.19 ng/mL vs. 3.51 ± 0.50 ng/mL, p < 0.05), and were suppressed by atorvastatin administration. Correlation analysis showed high correlation between plasma APE1/Ref-1 levels and NLR, a marker of systemic inflammation. The cut-off value for APE1/Ref-1 for predicting atherosclerotic inflammation at 4.903 ng/mL showed sensitivity of 100% and specificity of 91%. We conclude that APE1/Ref-1 expression is upregulated in aortic endothelial cells/macrophages of atherosclerotic mice, and that plasma APE1/Ref-1 levels could predict atherosclerotic inflammation.
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BACKGROUND: Osteoarthritis (OA) is the most common type of joint disease associated with cartilage breakdown. However, the role played by mitochondrial dysfunction in OA remains inadequately understood. Therefore, we investigated the role played by p66shc during oxidative damage and mitochondrial dysfunction in OA and the effects of p66shc downregulation on OA progression. METHODS: Monosodium iodoacetate (MIA), which is commonly used to generate OA animal models, inhibits glycolysis and biosynthetic processes in chondrocytes, eventually causing cell death. To observe the effects of MIA and poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles, histological analysis, immunohistochemistry, micro-CT, mechanical paw withdrawal thresholds, quantitative PCR, and measurement of oxygen consumption rate and extracellular acidification rate were conducted. RESULTS: p-p66shc was highly expressed in cartilage from OA patients and rats with MIA-induced OA. MIA caused mitochondrial dysfunction and reactive oxygen species (ROS) production, and the inhibition of p66shc phosphorylation attenuated MIA-induced ROS production in human chondrocytes. Inhibition of p66shc by PLGA-based nanoparticles-delivered siRNA ameliorated pain behavior, cartilage damage, and inflammatory cytokine production in the knee joints of MIA-induced OA rats. CONCLUSION: p66shc is involved in cartilage degeneration in OA. By delivering p66shc-siRNA-loaded nanoparticles into the knee joints with OA, mitochondrial dysfunction-induced cartilage damage can be significantly decreased. Thus, p66shc siRNA PLGA nanoparticles may be a promising option for the treatment of OA.
Assuntos
Mitocôndrias/patologia , Osteoartrite/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Animais , Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Ácido Iodoacético/toxicidade , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/uso terapêutico , Osteoartrite/induzido quimicamente , Osteoartrite/patologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , RNA Interferente Pequeno/administração & dosagem , Ratos Sprague-DawleyRESUMO
The therapeutic benefits of repetitive transcranial magnetic stimulation (rTMS) combined with rehabilitation therapy on recovery after stroke have not been fully elucidated. This study aimed to explore the therapeutic effects of rTMS followed by aerobic exercise on neuroplasticity and recovery of motor function in a rat model of permanent middle cerebral artery occlusion (MCAO). Rats were randomized into sham operation (N = 10, sham op), MCAO (N = 10, control group), rTMS (N = 10, MCAO and rTMS therapy), and combination groups (N = 10, MCAO and combination therapy). High-frequency rTMS (10 Hz) was applied on the ipsilesional forepaw motor cortex, and aerobic exercise training on the rotarod was performed for two weeks. The rotarod and Garcia tests were conducted to evaluate changes in behavioral function. Motor evoked potentials (MEPs) were used to evaluate electrophysiological changes. Stroke severity was assessed using infarction volume measurement. Neuronal recovery was explored with western blot for brain-derived neurotrophic factor (BDNF) pathway proteins. Compared with control therapy, combination therapy was significantly more effective than rTMS therapy for improving function on the rotarod test (p = 0.08), Garcia test (p = 0.001), and MEP amplitude (p = 0.001) In conclusion, combination therapy may be a potential treatment to promote recovery of motor function and neuroplasticity in stroke patients.