RESUMO
ARVs alter the methylation status of the MEKKK1 gene promoter in acute treated Jurkat T cells with inflammatory outcomesInflammation is reduced in patients under going antiretroviral therapy; however the mechanism is not well understood. We investigated DNA methylation of the mitogen-activated protein kinase kinase kinase kinase 1 (MEKKK1) gene promoter in Jurkat T cells to determine whether the antiretroviral drugs, lamivudine, tenofovir disoproxil fumarate, dolutegravir, TLD (a combination of TDF, 3TC and DTG) and efavirenz modify the methylation status of the MEKKK1 gene - a known stimulus of inflammation.Acute antiretroviral treatments (24 h) were not cytotoxic to Jurkat T cells. MEKKK1 promoter hypomethylation occurred in cells treated with 5-aza-2'-deoxycytidine (Aza), TDF and 3TC, and MEKKK1 promoter hypermethylation occurred in cells treated with DTG; however, promoter DNA methylation of the MEKKK1 gene did not influence MEKKK1 gene expression; therefore, these drugs did not epigenetically regulate MEKKK1 and downstream signalling by promoter DNA methylation. Acute TLD and EFV treatments induced inflammation in Jurkat T cells by increasing MEKKK1, MAPK/ERK and NFκB expression, and activating tumour necrosis factor-α (TNF-α) expression. ARVs decreased IL-10 gene expression, showing no anti-inflammatory activity.The data shows that the inflammation caused by ARVs is not related to the methylation status of MEKKK1 gene promoter and suggests an alternative stimulus via post-transcriptional/post-translational modifications may activate the canonical MEKKK1/NFκB pathway that leads to inflammation. Finally, an increase in NFκB activity and pro-inflammatory cytokine activation seemed to occur via the MAPK/ERK pathway following ARV treatments in Jurkat T cells.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Humanos , Citocinas , Infecções por HIV/tratamento farmacológico , Lamivudina/toxicidade , Lamivudina/uso terapêutico , Tenofovir/uso terapêutico , Benzoxazinas , Decitabina/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/induzido quimicamente , Linfócitos TRESUMO
BACKGROUND: HIV endemic populations are displaying higher incidence of metabolic disorders. HIV and the standard treatment are both associated with altered lipid and cholesterol metabolism, however gallstone disease (a cholesterol related disorder) in Sub-Saharan African populations is rarely investigated. METHODS: This study sought to evaluate hepatic expression of key genes in cholesterol metabolism (LDLr, HMGCR, ABCA1) and transcriptional regulators of these genes (microRNA-148a, SREBP2) in HIV positive patients on antiretroviral therapy presenting with gallstones. Liver biopsies from HIV positive patients (cases: n = 5) and HIV negative patients (controls: n = 5) were analysed for miR-148a and mRNA expression using quantitative PCR. RESULTS: Circulating total cholesterol was elevated in the HIV positive group with significantly elevated LDL-c levels(3.16 ± 0.64 mmol/L) relative to uninfected controls (2.10 ± 0.74 mmol/L; p = 0.04). A scavenging receptor for LDL-c, LDLr was significantly decreased (0.18-fold) in this group, possibly contributing to higher LDL-c levels. Transcriptional regulator of LDLr, SREBP2 was also significantly lower (0.13-fold) in HIV positive patients. Regulatory microRNA, miR-148a-3p, was reduced in HIV positive patients (0.39-fold) with a concomitant increase in target ABCA1 (1.5-fold), which regulates cholesterol efflux. CONCLUSIONS: Collectively these results show that HIV patients on antiretroviral therapy display altered hepatic regulation of cholesterol metabolizing genes, reducing cholesterol scavenging, and increasing cholesterol efflux.
Assuntos
Colelitíase/metabolismo , Colesterol/metabolismo , Infecções por HIV/metabolismo , Metabolismo dos Lipídeos/genética , Lipoproteínas LDL/sangue , Fígado/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Adulto , Fármacos Anti-HIV/uso terapêutico , Colelitíase/tratamento farmacológico , Colelitíase/etiologia , Colelitíase/genética , Feminino , Regulação da Expressão Gênica , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
PURPOSE: Studies have reported that erythritol, a low or non-glycemic sugar alcohol possesses anti-hyperglycemic and anti-diabetic potentials but the underlying mode of actions is not clear. This study investigated the underlying mode of actions behind the anti-hyperglycemic and anti-diabetic potentials of erythritol using different experimental models (experiment 1, 2 and 3). METHODS: Experiment 1 examined the effects of increasing concentrations (2.5-20%) of erythritol on glucose absorption and uptake in isolated rat jejunum and psoas muscle, respectively. Experiments 2 and 3 examined the effects of a single oral dose of erythritol (1 g/kg bw) on intestinal glucose absorption, gastric emptying and postprandial blood glucose increase, glucose tolerance, serum insulin level, muscle/liver hexokinase and liver glucose-6 phosphatase activities, liver and muscle glycogen contents and mRNA and protein expression of muscle Glut-4 and IRS-1 in normal and type 2 diabetic animals. RESULTS: Experiment 1 revealed that erythritol dose dependently enhanced muscle glucose ex vivo. Experiment 2 demonstrated that erythritol feeding delayed gastric emptying and reduced small intestinal glucose absorption as well as postprandial blood glucose rise, especially in diabetic animals. Experiment 3 showed that erythritol feeding improved glucose tolerance, muscle/liver hexokinase and liver glucose-6 phosphatase activities, glycogen storage and also modulated expression of muscle Glut-4 and IRS-1 in diabetic animals. CONCLUSION: Data suggest that erythritol may exert anti-hyperglycemic effects not only via reducing small intestinal glucose absorption, but also by increasing muscle glucose uptake, improving glucose metabolic enzymes activity and modulating muscle Glut-4 and IRS-1 mRNA and protein expression. Hence, erythritol may be a useful dietary supplement for managing hyperglycemia, particularly for T2D.
Assuntos
Glicemia/metabolismo , Eritritol/farmacologia , Absorção Gastrointestinal/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Músculo Esquelético/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Glucose , Transportador de Glucose Tipo 4/metabolismo , Insulina , Ratos , Ratos Sprague-DawleyRESUMO
Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG2 cells with FA (IC50 : 580.32 µM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Carcinoma Hepatocelular/metabolismo , Dano ao DNA , Ácido Fusárico/farmacologia , Neoplasias Hepáticas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologiaRESUMO
The ATP-binding cassette (ABC) super-family of drug transporters regulates efflux of xenobiotic compounds. The subfamily, multi-drug resistance proteins (MRPs) transports cyclic nucleotides and xenobiotics. Epigenetic modulation of drug transporters is scarcely described. The regulatory role of microRNA (miR)-124a on drug transporter gene ABCC4 was only recently reported. Our study investigated the differential regulation of miR-124a by nucleoside reverse transcriptase inhibitors (NRTIs): Zidovudine (AZT), Stavudine (d4T) and Tenofovir (TFV); at 24 h and 120 h treatments in HepG2 cells. ABCC4 mRNA (qPCR) and ABCC4 protein (western blot) were quantified. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) levels. All NRTIs elevated miR-124a levels at 24 h, with a concomitant decline in ABCC4 mRNA levels (p<0.05). At 120 h, d4T and TFV elevated miR-124a and depleted ABCC4 mRNA levels (p<0.0001), while the inverse was observed with AZT (p<0.005). ABCC4 protein was increased by d4T and TFV at 24h. A significant reduction in protein levels was observed at 120 h in all three treatments (p<0.005). The disjoint in mRNA and protein levels is likely due to ABCC4 being a membrane bound protein. Following prolonged exposure, membrane integrity was compromised as evidenced by increased LDH leakage (p<0.005). We conclude antiretroviral drugs have varying effects on miR-124a and ABCC4.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antirretrovirais/farmacologia , MicroRNAs/metabolismo , Células Hep G2 , Humanos , Tenofovir , ZidovudinaRESUMO
Chronic HIV treatment with antiretroviral drugs has been associated with adverse health outcomes. Mitochondrial toxicity exhibited by nucleoside reverse transcriptase inhibitors (NRTIs) is pinpointed as a molecular mechanism of toxicity. This study evaluated the effect of NRTIs: Zidovudine (AZT, 7.1 µM), Stavudine (d4T, 4 µM) and Tenofovir (TFV, 1.2 µM), on mitochondrial (mt) stress response, mtDNA integrity and oxidative stress response in human hepatoma cells at 24 and 120 h. Markers for mt function, mt biogenesis, oxidative stress parameters, and antioxidant response were evaluated by spectrophotometry, luminometry, flow cytometry, qPCR and western blots. We found that AZT and d4T reduced mtDNA integrity (120 h, AZT: 76.1%; d4T:36.1%, P < 0.05) and remained unchanged with TFV. All three NRTIs, however, reduced ATP levels (AZT: 38%; d4T: 56.4%; TFV: 27.4%, P = 0.01) and mt membrane potential at 120 h (P < 0.005). Oxidative damage and reactive oxygen species (ROS) were increased by TFV and AZT at 24 h, and by d4T at 120 h (P < 0.05). Antioxidant response molecules and mt biogenesis markers were elevated by all NRTIs, with TFV causing the most significant increase (P < 0.05). Data from this study suggest that AZT, d4T and TFV alter mt function. TFV, however, achieves this independently of mtDNA depletion. Furthermore, AZT exerts toxicity soon after exposure as noted from changes at 24 h and d4T exerts greater toxicity over prolonged exposure (120 h).
Assuntos
Antirretrovirais/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/genética , Espécies Reativas de Oxigênio/metabolismo , Estavudina/toxicidade , Tenofovir/toxicidade , Zidovudina/toxicidadeRESUMO
Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by Aspergillus and Penicillium fungi. It contaminates human and animal food products, and chronic exposure is associated with renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol, a phytoalexin, possesses anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA, and the effect of resveratrol in human embryonic kidney (HEK293) cells over 24 and 48 h. Cells were exposed to OTA [IC50 = 1.5 µM (24 h) and 9.4 µM (48 h) determined using MTT assay] and 25 µM resveratrol. Glutathione was quantified by luminometry and gene expression of Nrf2 and OGG1 was determined by qPCR. Protein expression of Nrf2, LonP1, SIRT3, and pSIRT1 was assessed by Western blot, DNA damage (comet assay), and intracellular reactive oxygen species (flow cytometry). At 24 h, resveratrol increased mRNA expression of the DNA repair enzyme, OGG1 (P < 0.05), whereas OTA and OTA+resveratrol significantly decreased OGG1 expression (P < 0.05). OGG1 expression increased during 48-h exposure to resveratrol and OTA+resveratrol (P < 0.05). Comet tail lengths doubled in 48-h OTA-treated cells, whereas at both time periods, OTA+resveratrol yielded shorter comet tails (P < 0.0001). During 24- and 48-h exposure, OTA, resveratrol, and OTA+resveratrol significantly decreased mRNA expression of Nrf2 (P < 0.05). Luminometry analysis of GSH revealed an increase by OTA+resveratrol for 24 and 48 h (P < 0.05 and P < 0.001, respectively). Western blot analysis showed decreased Nrf2 protein expression during 24-h exposure, but increased Nrf2 expression during 48 h. LonP1 protein expression increased during 24-h exposure to OTA (P < 0.05) and OTA+resveratrol (P < 0.0011) and during 48-h exposure to resveratrol (P < 0.0005).
Assuntos
Nefropatia dos Bálcãs/tratamento farmacológico , Ocratoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estilbenos/administração & dosagem , Proteases Dependentes de ATP/biossíntese , Apoptose/efeitos dos fármacos , Aspergillus/patogenicidade , Nefropatia dos Bálcãs/patologia , Dano ao DNA , DNA Glicosilases/biossíntese , Microbiologia de Alimentos , Células HEK293 , Humanos , Proteínas Mitocondriais/biossíntese , Penicillium/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Sesquiterpenos/administração & dosagem , FitoalexinasRESUMO
Non-communicable diseases (NCDs) have risen rapidly worldwide, sparking interest in causative agents and pathways. Patulin (PAT), a xenobiotic found in fruit products contaminated by molds, is postulated to be diabetogenic in animals, but little is known about these effects in humans. This study examined the effects of PAT on the insulin signaling pathway and the pyruvate dehydrogenase complex (PDH). HEK293 and HepG2 cells were exposed to normal (5 mM) or high (25 mM) glucose levels, insulin (1.7 nM) and PAT (0.2 µM; 2.0 µM) for 24 h. The qPCR determined gene expression of key enzymes involved in carbohydrate metabolism while Western blotting assessed the effects of PAT on the insulin signaling pathway and Pyruvate Dehydrogenase (PDH) axis. Under hyperglycemic conditions, PAT stimulated glucose production pathways, caused defects in the insulin signaling pathway and impaired PDH activity. These trends under hyperglycemic conditions remained consistent in the presence of insulin. These findings are of importance, given that PAT is ingested with fruit and fruit products. Results suggest PAT exposure may be an initiating event in insulin resistance, alluding to an etiological role in the pathogenesis of type 2 diabetes and disorders of metabolism. This highlights the importance of both diet and food quality in addressing the causes of NCDs.
Assuntos
Diabetes Mellitus Tipo 2 , Patulina , Humanos , Animais , Patulina/toxicidade , Células HEK293 , Insulina , Transdução de SinaisRESUMO
Female sex, high estrogen levels, aging, obesity, and dyslipidemia are some of the risk factors associated with gallstone formation. HIV-infected patients on combination antiretroviral therapy (cART) are more prone to hypercholesterolemia. Bile acid synthesis is initiated by cholesterol 7-alpha hydroxylase (CYP7A1) and regulated by hepatocyte nuclear factors (HNF1α, HNF4α, and LXRb). The aim of this study was to evaluate the expression of HNF1α, HNF4α, LXRb, and miRNAs (HNF4α specific: miR-194-5p and miR-122*_1) that regulate CYP7A1 transcription in HIV-infected Black South African women on cART and presenting with gallstones relative to HIV-negative patients with gallstone disease. Females (n = 96) presenting with gallstone disease were stratified based on HIV status. The gene expression of CYP7A1, HNF1α, HNF4α, LXRb, miR-194-5p, and miR-122*_1 was determined using RT-qPCR. Messenger RNA and miRNA levels were reported as fold change expressed as 2-ΔΔCt (RQ min; RQ max). Fold changes >2 and <0.5 were considered significant. HIV-infected females were older in age (p = 0.0267) and displayed higher low-density lipoprotein cholesterol (LDL-c) (p = 0.0419), CYP7A1 [2.078-fold (RQ min: 1.278; RQ max: 3.381)], LXRb [2.595-fold (RQ min: 2.001; RQ max: 3.000)], and HNF1α [3.428 (RQ min: 1.806; RQ max: 6.507] levels. HNF4α [0.642-fold (RQ min: 0.266; RQ max: 1.55)], miR-194-5p [0.527-fold (RQ min: 0.37; RQ max: 0.752)], and miR-122*_1 [0.595-fold (RQ min: 0.332; RQ max: 1.066)] levels were lower in HIV-infected females. In conclusion, HIV-infected women with gallstone disease displayed higher LDL-c levels and increased bile acid synthesis, which was evidenced by the elevated expression of CYP7A1, HNF1α, and LXRb. This could have been further influenced by cART and aging.
RESUMO
N6-methyladenosine (m6A) is an abundant epitranscriptomic mark that regulates gene expression to execute cellular developmental programmes and environmental adaptation. Fusaric acid (FA) is a mycotoxin that contaminates agricultural foods and exerts toxicity in humans and animals; however, its epitranscriptomic effects are unclear. We investigated the effect of FA on global m6A RNA methylation and mRNA expression levels of key m6A regulatory genes in C57BL/6 mouse livers. C57BL/6 mice (n = 6/group) were orally administered 0.1 M phosphate-buffered saline (PBS) or 50 mg/kg FA. Mice were euthanized 24 h after oral administration, livers were harvested, and RNA was isolated. RNA samples were assayed for global m6A levels using an m6A RNA Methylation Quantification Kit. The mRNA expression of m6A regulators i.e. writers, erasers, and readers were measured by qRT-PCR. FA increased global m6A RNA methylation (p < 0.0001) in mouse livers. FA increased the expression of METTL3 (p = 0.0143) and METTL14 (p = 0.0281), and decreased the expression of FTO (p = 0.0036) and ALKBH5 (p = 0.0035). The expression of YTHDF2 (p = 0.0007), YTHDF3 (p = 0.0061), and YTHDC2 (p = 0.0258) were increased by FA in mouse livers. This study shows that the liver m6A epitranscriptome can be modified by FA exposure in an in vivo model and can be useful for identifying the molecular mechanisms whereby m6A RNA modifications influence the toxicological outcomes of FA exposure.
Assuntos
Metilação de DNA , Ácido Fusárico , Animais , Genes Reguladores , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Projetos Piloto , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The introduction of highly active antiretroviral therapy (HAART) in the treatment of HIV/AIDS has recently gained popularity. In addition, the significant role of microRNA expression in HIV pathogenesis cannot be overlooked; hence the need to explore the mechanisms of microRNA expression in the presence of HAART and Spirulina platensis (SP) in HepG2 cells. This study investigates the biochemical mechanisms of microRNA expression in HepG2 cells in the presence of HAART, SP, and the potential synergistic effect of HAART−SP. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability following SP treatment. The cellular redox status was assessed using the quantification of intracellular reactive oxygen species (ROS), lipid peroxidation, and a lactate dehydrogenase (LDH) assay. The fluorometric JC-1 assay was used to determine mitochondrial polarisation. The quantitative polymerase chain reaction (qPCR) was also employed for micro-RNA and gene expressions. The results show that MiR-146a (p < 0.0001) and miR-155 (p < 0.0001) levels increased in SP-treated cells. However, only miR-146a (p < 0.0001) in HAART−SP indicated an increase, while miR-155 (p < 0.0001) in HAART−SP treatment indicated a significant decreased expression. Further inflammation analysis revealed that Cox-1 mRNA expression was reduced in SP-treated cells (p = 0.4129). However, Cox-1 expression was significantly increased in HAART−SP-treated cells (p < 0.0001). The investigation revealed that HepG2 cells exposed to HAART−SP treatment showed a significant decrease in Cox-2 (p < 0.0001) expression. mRNA expression also decreased in SP-treated cells (p < 0.0001); therefore, SP potentially controls inflammation by regulating microRNA expressions. Moreover, the positive synergistic effect is indicated by normalised intracellular ROS levels (p < 0.0001) in the HAART−SP treatment. We hereby recommend further investigation on the synergistic roles of SP and HAART in the expression of microRNA with more focus on inflammatory and oxidative pathways.
RESUMO
Patulin (PAT) is a mycotoxin produced by Penicillium and other fungi that contaminate fruit. PAT targets the kidney and is associated with nephrotoxicity. Micro-RNAs (miRNA) may offer new insights into PAT-induced nephrotoxicity. Cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1), involved in metabolism of dietary toxins is negatively regulated by miR-27b and linked with the nuclear factor kappa B (NF-κB) pathway and peroxisome proliferator activated receptor gamma (PPARÉ£) in renal fibrosis. This study investigated the effects of PAT on miR-27b, CYP1B1, PPARÉ£ and cytotoxicity in human kidney (HEK293) cells. HEK293 cells were exposed to PAT (2.5 µM, 24h). Protein expression of CYP1B1, PPARÉ£, NF-κB (p65), pNF-κB (p65) (phospho-Ser563) and cleaved PARP-1 was quantified using western blotting. QPCR evaluated mRNA levels of CYP1B1, IL-6, miR-27b, OGG1, mtDNA, TFAM and UCP2. Mitochondrial membrane potential and phosphatidylserine (PS) externalization was evaluated by flow cytometry while levels of ATP and caspase -9, -8, -3/7 activity was measured using luminometry. PAT significantly decreased miR-27b levels (p = 0.0014) and increased CYP1B1 mRNA (p = 0.0015) and protein (p = 0.0013) levels. PPARÉ£ protein expression was significantly increased (p = 0.0002) and associated with decreased NF-κB activation (p = 0.0273) and IL-6 mRNA levels (p = 0.0265). Finally, PAT significantly compromised mitochondrial repair mechanisms and increased apoptotic biomarkers. PAT altered miR-27b levels and PPARÉ£, with associated changes to NF-κB activation, downstream IL-6 and CYP1B1 expression. These results show that PAT impairs detoxification mechanisms leading to mitochondrial damage and apoptosis. In conclusion, PAT altered the epigenetic environment and impaired detoxification processes, supporting a mechanism for nephrotoxic outcomes.
Assuntos
MicroRNAs , Patulina , Células HEK293 , Humanos , MicroRNAs/genética , NF-kappa B/metabolismo , Patulina/toxicidade , RNA Mensageiro/metabolismoRESUMO
Lately, Spirulina platensis (SP), as an antioxidant, has exhibited high potency in the treatment of oxidative stress, diabetes, immune disorder, inflammatory stress, and bacterial and viral-related diseases. This study investigated the possible protective role of Spirulina platensis against ARV-induced oxidative stress in HepG2 cells. Human liver (HepG2) cells were treated with ARVs ((Lamivudine (3TC): 1.51 µg/mL, tenofovir disoproxil fumarate (TDF): 0.3 µg/mL and Emtricitabine (FTC): 1.8 µg/mL)) for 96 h and thereafter treated with 1.5 µg/mL Spirulina platensis for 24 h. After the treatments, the gene and protein expressions of the antioxidant response pathway were determined using a quantitative polymerase chain reaction (qPCR) and Western blots. The results show that Spirulina platensis decreased the gene expressions of Akt (p < 0.0001) and eNOS (↓p < 0.0001) while, on the contrary, it increased the transcript levels of NRF-2 (↑p = 0.0021), Keap1 (↑p = 0.0002), CAT (↑p < 0.0001), and NQO-1 (↑p = 0.1432) in the HepG2 cells. Furthermore, the results show that Spirulina platensis also decreased the protein expressions of NRF-2 (↓p = 0.1226) and pNRF-2 (↓p = 0.0203). Interestingly, HAART-SP induced an NRF-2 pathway response through upregulating NRF-2 (except for FTC-SP) (↑p < 0.0001), CAT (↑p < 0.0001), and NQO-1 (except for FTC-SP) (↑p < 0.0001) mRNA expression. In addition, NRF-2 (↑p = 0.0085) and pNRF-2 (↑p < 0.0001) protein expression was upregulated in the HepG2 cells post-exposure to HAART-SP. The results, therefore, allude to the fact that Spirulina platensis has the potential to mitigate HAART-adverse drug reactions (HAART toxicity) through the activation of antioxidant response in HepG2 cells. We hereby recommend further studies on Spirulina platensis and HAART synergy.
RESUMO
Ubiquitous soil fungi parasitise agricultural commodities and produce mycotoxins. Fumonisin B2 (FB2), the structural analogue of the commonly studied Fumonisin B1 (FB1), is a neglected mycotoxin produced by several Fusarium species. Mycotoxins are known for inducing toxicity via mitochondrial stress alluding to mitochondrial degradation (mitophagy). These processes involve inter-related pathways that are regulated by proteins related to SIRT3 and Nrf2. This study aimed to investigate mitochondrial stress responses in human kidney (Hek293) cells exposed to FB2 for 24 h. Cell viability was assessed via the methylthiazol tetrazolium (MTT) assay, and the half-maximal inhibitory concentration (IC50 = 317.4 µmol/L) was estimated using statistical software. Reactive oxygen species (ROS; H2DCFDA), mitochondrial membrane depolarisation (JC1-mitoscreen) and adenosine triphosphate (ATP; luminometry) levels were evaluated to assess mitochondrial integrity. The relative expression of mitochondrial stress response proteins (SIRT3, pNrf2, LONP1, PINK1, p62 and HSP60) was determined by Western blot. Transcript levels of SIRT3, PINK1 and miR-27b were assessed using quantitative PCR (qPCR). FB2 reduced ATP production (p = 0.0040), increased mitochondrial stress marker HSP60 (p = 0.0140) and suppressed upregulation of mitochondrial stress response proteins SIRT3 (p = 0.0026) and LONP1 (p = 0.5934). FB2 promoted mitophagy via upregulation of pNrf2 (p = 0.0008), PINK1 (p = 0.0014) and p62 (p < 0.0001) protein expression. FB2 also suppressed miR-27b expression (p < 0.0001), further promoting the occurrence of mitophagy. Overall, the findings suggest that FB2 increases mitochondrial stress and promotes mitophagy in Hek293 cells.
Assuntos
Fumonisinas , MicroRNAs , Micotoxinas , Sirtuína 3 , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Fumonisinas/toxicidade , Células HEK293 , Humanos , Rim/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia , Proteínas Quinases , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
The brain responds to diabetic stress by inducing the inflammatory response. Under normal circumstances this process is tightly regulated. However, uncontrolled inflammatory responses lead to compromised function and eventual neurodegeneration. The microRNA (miR)-200 family, specifically miR-141, is differentially expressed in diseased states including cognitive decline, thereby triggering changes in downstream genes. We hypothesised that Metformin (MF) regulates the miR-141/protein phosphatase 2A (PP2A) axis, and associated NF-ĸB-mediated inflammasome expression in diabetic mice brain. Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ), thereafter mice were treated with MF (20 mg/kg BW). Whole brain tissue was harvested for further analysis. In silico analysis showed that Sirt1 and PP2A are prediction targets of miR-141. Selected protein and gene expressions were established through western blotting and qPCR, respectively. Diabetic mice brain tissue demonstrated overexpression of miR-141 and related pro-inflammatory factors as well as decreased PP2A gene expression. MF was able to counteract this by regulating expression of miR-141, PP2A, and p-tau at Ser396 protein expressions. Further experimentation revealed MF's inhibitory action on the inflammasome system by regulating the expression of the upstream controller NLRP3, related cytokines and NF-κB signalling pathway. Collectively, we demonstrate that MF promotes neuroprotection in diabetic mice by dampening inflammatory responses through its inhibitory effects on various signalling pathways. CATEGORIES: Inflammation and Immunopharmacology, Metabolic Disorders and Endocrinology, Neuropharmacology.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Inflamassomos/metabolismo , Inflamação/prevenção & controle , Metformina/farmacologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Fármacos Neuroprotetores/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Biologia Computacional , Diabetes Mellitus Experimental/metabolismo , Inflamação/metabolismo , Masculino , Metformina/uso terapêutico , Camundongos Endogâmicos C57BL , MicroRNAs/genética , NF-kappa B/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/genética , Sirtuína 1/metabolismo , Estreptozocina , Proteínas tau/metabolismoRESUMO
Fusaric acid (FA) is a food-borne mycotoxin that mediates toxicity with limited information on its epigenetic properties. p53 is a tumour suppressor protein that regulates cell cycle arrest and apoptotic cell death. The expression of p53 is regulated transcriptionally by promoter methylation and post-transcriptionally by N-6-methyladenosine (m6A) RNA methylation. We investigated the effect of FA on p53 expression and its epigenetic regulation via promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells. HepG2 cells were treated with FA [0, 25, 50, 104, and 150 µg/ml; 24 h] and thereafter, DNA, RNA, and protein was isolated. Promoter methylation and expression of p53 was measured using qPCR and Western blot. RNA immuno-precipitation was used to determine m6A-p53 levels. The expression of m6A methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), and readers (YTHDF1-3 and YTHDC2) were measured using qPCR. FA induced p53 promoter hypermethylation (p < 0.0001) and decreased p53 expression (p < 0.0001). FA decreased m6A-p53 levels (p < 0.0001) by decreasing METTL3 (p < 0.0001) and METTL14 (p < 0.0001); and suppressed expression of YTHDF1 (p < 0.0001), YTHDF3 (p < 0.0001), and YTHDC2 (p < 0.0001) that ultimately reduced p53 translation (p < 0.0001). Taken together, the data shows that FA epigenetically decreased p53 expression by altering its promoter methylation and m6A RNA methylation in HepG2 cells. This study reveals a mechanism for p53 regulation by FA and provides insight into future therapeutic interventions.
Assuntos
Metilação de DNA/efeitos dos fármacos , Ácido Fusárico/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Células Hep G2 , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Regiões Promotoras Genéticas , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: The incidence of metabolic disorders in human immunodeficiency virus (HIV) endemic settings is a prevailing burden in developing countries. Cholesterol homeostasis and fat metabolism are altered by HIV and antiretroviral therapy (ART), thereby possibly contributing to complications such as gallstone formation. OBJECTIVES: The aim of this study was to evaluate established risk factors for the formation of cholesterol gallstones in black South African women living with HIV (WLHIV). METHOD: A case series study was conducted of all black South African women undergoing cholecystectomy for gallstone disease over a 1-year period at King Edward VIII Hospital, Durban, South Africa. Age, body mass index (BMI), family history of gallstones, oestrogen exposure and lipograms were compared between WLHIV and uninfected women. Categorical variables were tested using either the Fisher's exact test or Pearson's chi-square test. Means were compared using independent t-tests. For non-normally distributed data, the Mann-Whitney U test was used. Statistical tests were two-sided, and p-values of less than 0.05 were considered statistically significant. RESULTS: A total of 52 patients were assessed, 34 HIV-uninfected and 18 WLHIV. The median age of WLHIV versus the uninfected women was 35 and 50 years, respectively, (p = 0.015). A statistically significant number of uninfected women were in the overweight/obese category (BMI > 25 kg/m2) compared to the normal weight category (BMI < 25 kg/m2) (p < 0.001). The number of obese WLHIV did not reach statistical significance. CONCLUSION: The age of occurrence of gallstone disease amongst black South African WLHIV was significantly lower and fewer women were obese compared with the uninfected women with gallstone disease. These findings differ from known gallstone risk factors in other populations and in uninfected black South African women. This could be attributed to the metabolic alterations caused by HIV infection itself and/or to the long-term use of ART. Larger cohort studies are required to elucidate the role of HIV and ART in cholestatic disease.
RESUMO
Patulin (PAT) is a mycotoxin produced by various fungal species that commonly contaminate apples and other fruit products. PAT is associated with glutathione (GSH) depletion and oxidative stress. Cytoprotective and antioxidant (AO) enzymes limit toxic outcomes and confer resistance to oxidative stress by influencing the expression of cytoprotective genes. The induction of these genes is tightly regulated by transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2), a potential target of microRNA (miR)-144. This study aims to determine a possible role for miR-144 in NRF2 pathway activation following PAT exposure in human embryonic kidney (HEK293) cells. HEK293 cells were exposed to varying PAT concentrations (0, 0.2, 0.5, 1 µmol/L; 24 h). Protein expression of Keap1, NRF2, and phosphorylated (p) NRF2 (ser40) was quantified using western blotting. Gene expression of NRF2, SOD2, CAT, GPx, NQO1, GSTA1, HMOX, and miR-144 were evaluated by qPCR. PAT significantly decreased miR-144 (p = 0.0249) and concomitantly increased NRF2 protein expression, stability, and activation as evidenced by increased pNRF2 (p = 0.0216) expression and decreased total NRF2 (p = 0.0237). This was consistent with qPCR data which showed increased transcript levels of NRF2 (p = 0.0378) as well as the target genes CAT (p = 0.0273), NQO1 (p = 0.0156), HMOX (p = 0.0249), and GSTA1 (p = 0.0237). No changes were observed in Keap1 expression (p = 0.6444). This study implicates microRNAs in a mechanistic role in PAT-induced toxicity. PAT decreased miR-144 expression leading to NRF2 pathway activation and elevated AO gene expression.
Assuntos
Redes e Vias Metabólicas/efeitos dos fármacos , MicroRNAs/genética , Micotoxinas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Patulina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Expressão Gênica , Células HEK293 , HumanosRESUMO
Deoxynivalenol (DON) commonly infects agricultural foods; it exhibits toxicity by inducing oxidative stress and inhibiting protein synthesis. Nuclear factor erythroid 2-related factor 2 (NRF2) regulates the cellular antioxidant response. We investigated the cytotoxicity of DON and its effect on the NRF2 antioxidant response in HepG2 cells. The Methyl Thiazol Tetrazolium (MTT), glutathione (GSH) and ATP assays evaluated toxicity, whilst lipid peroxidation and membrane damage were assessed using the Thiobarbituric acid reactive substance (TBARS) and lactate dehydrogenase (LDH) assays. Protein expression of NRF2, phosphorylated (p-ser40) NRF2, catalase (CAT), superoxide dismutase 2 (SOD2), and Sirtuin 3 (Sirt3) were quantified by Western Blotting. Gene expression of glutathione peroxidase (GPx), CAT and SOD2 was determined using qPCR. DON decreased cell viability, GSH concentrations and ATP levels and increased lipid peroxidation and membrane damage. DON significantly decreased total NRF2 and increased p-NRF2 and downregulated the transcription and translation of NRF2 target antioxidant enzymes. Further, expression of the mitochondrial stress response protein, Sirt3 was significantly decreased. In conclusion, DON induced oxidative stress and downregulated NRF2-induced cytoprotection by suppressing the antioxidant signalling mechanism in HepG2 cells.
Assuntos
Fator 2 Relacionado a NF-E2/metabolismo , Tricotecenos/toxicidade , Antioxidantes , Carcinoma Hepatocelular , Células Hep G2 , Humanos , Neoplasias Hepáticas , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
Metabolic flexibility defines the capacity of cells to respond to changes in nutrient status. Mitochondria are important mediators of metabolic flexibility and dysfunction is associated with metabolic inflexibility and pathology. Foodborne toxins are often overlooked as potential factors contributing to metabolic toxicity. Fusaric acid (FA), a neglected mycotoxin, is known to disrupt mitochondrial function. The aim of this study was to investigate the molecular mechanisms underlying a metabolic switch in response to FA. This study investigated the effects of FA on energy homeostasis in cultured human liver (HepG2) cells. HepG2 cells poised to undergo oxidative and glycolytic metabolism were exposed to a range of FA concentrations (4, 63 and 250⯵g/mL) for 6â¯h. We determined mitochondrial toxicity, acetyl CoA levels and cell viability using luminometric, fluorometric and spectrophotometric methods. Expression of metabolic proteins (PDK1, PKM2, phosphorylated-PDH E1α and HIF-1α) and mRNAs (HIF-1α, PKM2, LDHa and PDK1) were determined using western blot and qPCR respectively. Our data connects a constitutive expression of HIF-1α in response to FA, to the inhibition of pyruvate decarboxylation through up-regulation of PDK-1 and phosphorylation of Pyruvate Dehydrogenase E1α subunit. Moreover, we highlight the potential of FA to induce a glucose "addiction" and phenotype reminiscent of the Warburg effect. The findings provide novel insights into the impact of this neglected foodborne mycotoxin in the dysregulation of energy metabolism.