Efeitos da administração de sulfito sobre a estrutura da mielina e inflamação em estriado de ratos jovens

A deficiência da sulfito oxidase (SO) é um erro inato do metabolismo de herança autossômica causada pela deficiência isolada da enzima ou por defeitos na síntese do cofator molibdênio. A doença é bioquimicamente caracterizada pelo acúmulo tecidual e elevada excreção urinária predominantemente de sulfito. Os pacientes apresentam convulsões graves, anormalidades nos gânglios basais e desmielinização que frequentemente causam óbito nos primeiros meses de vida. Visto que a etiopatogenia do dano neurológico observado na deficiência da SO não está esclarecida, avaliamos os efeitos da administração intraestriatal de sulfito (2 mol) sobre parâmetros de estrutura e compactação da mielina e de inflamação em estriado de ratos jovens eutanasiados 7 dias após a administração. Além disso, visando contribuir para o desenvolvimento de novas estratégias terapêuticas para essa doença, avaliamos a influência da administração por gavagem de bezafibrato (BEZ; 30 ou 100 mg/kg/dia) realizada antes (pré-tratamento) ou depois (pós-tratamento) da injeção de sulfito. A administração de sulfito diminuiu os feixes de axônios no estriado, observado através da marcação da Fluoromielina, uma sonda com alta sensibilidade que marca áreas mielinizadas, e também a intensidade da marcação da proteína básica de mielina (MBP), um componente integral da mielina. O sulfito ainda aumentou a marcação de neuroglicano-2 (NG2), um marcador de células precursoras de oligodendrócitos, indicando indução de remielinização. Tanto o pré- quanto o pós-tratamento com BEZ atenuaram ou preveniram a diminuição dos feixes de axônios e a marcação da MBP, ao passo que o pós-tratamento preveniu o aumento da marcação do NG2. Além disso, foi observado que o sulfito aumentou a intensidade da marcação da molécula adaptadora ligante de cálcio ionizado-1 (Iba1), e que o pós-tratamento com BEZ preveniu esse efeito. No que se refere aos parâmetros indicativos de processo inflamatório, o sulfito aumentou os níveis do RNAm do fator de transcrição NFkB, porém não modificou os níveis do RNAm das citocinas fator de necrose tumoral e interleucina-1. Portanto, nossos resultados demonstram que o sulfito causa alterações na mielina e induz inflamação em estriado. Pode ser presumido que tais mecanismos patológicos contribuem, ao menos em parte, para as anormalidades e desmielinização dos gânglios basais observadas em pacientes com deficiência da SO. Já o fato de o BEZ ter prevenido alguns dos efeitos tóxicos do sulfito sugere que esse composto pode ser um potencial adjuvante a ser usado na terapia para a deficiência da SO.Sulfite oxidase (SO) deficiency is an autosomal recessive inborn error of metabolism caused either by the isolated deficiency of SO or by defects in the molybdenum cofactor synthesis. SO deficiency is biochemically characterized by predominant tissue accumulation and high urinary excretion of sulfite. Patients usually present severe seizures, basal ganglia abnormalities and demyelination that often lead to early death. Considering that the etiopathogenesis of the neurological dysfunction observed in SO deficiency is not totally established, we evaluated the effects of intrastriatal administration of sulfite on parameters of myelin compaction, structure, and inflammation in striatum of young rats euthanized 7 days after sulfite injection. Aiming to contribute for the development of novel therapeutic strategies for this disorder, we also evaluated the influence of bezafibrate (BEZ) administration (30 or 100 mg/kg/day) performed by gavage before (pre-treatment) or after (post-treatment) sulfite injection. Our results demonstrated that sulfite administration reduced axonal bundles in striatum, as observed with the Fluoromyelin probe that detects myelinating areas with high sensitivity. Sulfite also decreased the intensity of myelin basic protein (MBP) staining, an integral component of myelin, and increased neuroglycan-2 (NG2) staining, a marker of oligodendrocyte precursor cells. In addition, BEZ pre- and post-treatment prevented or attenuated the decrease of axonal bundles and MBP staining intensity caused by sulfite, whereas BEZ post-treatment prevented NG2 increase. Regarding the inflammation parameters, sulfite significantly increased the staining of ionized calcium-binding adapter molecule 1 (Iba1) while BEZ post-treatment prevented this effect. Furthermore, sulfite increased mRNA levels of NFkB, but did not alter of the cytokines tumor necrosis factor and interleukin-1. Therefore, our findings showing that sulfite causes myelin alterations and inflammation in striatum of rats suggest that these pathomechanisms are involved, at least in part, in the abnormalities and demyelination observed in basal ganglia of SO deficient individuals. Moreover, since BEZ prevented some of the toxic effects exerted by sulfite, we suggest that this compound should be further investigated as a potential adjuvant therapy for SO deficiency

Estudo de mecanismos envolvidos na neurofisiopatologia da deficiência da sulfito oxidase em ratos neonatos e jovens

A deficiência da sulfito oxidase (SO) é uma condição de caráter autossômico recessivo que pode ser encontrada em duas formas: deficiência isolada da SO e deficiência do cofator molibdênio. A deficiência isolada da SO ocorre devido a mutações no gene SUOX, responsável por codificar a apoenzima da SO propriamente dita, levando à diminuição ou ausência de atividade, e posterior degradação da enzima. Já a segunda forma é causada pela deficiência na atividade de alguma das enzimas que participam da rota de biossíntese do cofator molibdênio. Visto que a enzima SO é responsável pela oxidação de sulfito a sulfato, ambas as formas são caracterizadas pelo acúmulo tecidual de sulfito e tiossulfato. Além disso, são clinicamente caracterizadas por convulsões neonatais graves, disfunção neurológica progressiva, hipotonia axial, hipertonicidade periférica e atraso no desenvolvimento psicomotor, o que comumente resulta em morte prematura. Considerando que a fisiopatologia do dano neurológico observado na deficiência da SO não está totalmente estabelecida, no primeiro capítulo desta tese foram investigados os efeitos in vivo do sulfito em estriado de ratos jovens sobre defesas antioxidantes, atividade da creatina cinase (CK), via das MAPK, e marcadores de apoptose. Também foi avaliada a influência do sequestrador de espécies reativas direcionado à mitocôndria JP4-039 sobre os efeitos tóxicos do sulfito. Para isso, ratos de 30 dias de vida receberam uma única dose intraestriatal de sulfito (Na2SO3; 2μmol; grupo teste) ou NaCl (2μmol; grupo controle), sendo eutanasiados 30 min após a injeção. Em outros grupos, os ratos receberam duas injeções intraperitoneais de JP4-039 (uma injeção de 3,5μg/g e outra de 5μg/g ou duas injeções de 5 μg/g), 24 e 2 horas antes da administração de sulfito ou NaCl. Após a eutanásia, o estriado foi removido e homogeneizado para as análises. O sulfito diminuiu as concentrações de glutationa reduzida (GSH) e as atividades das enzimas glutationa peroxidase (GPx), glucose-6-fosfato desidrogenase (G6PDH), glutationa redutase (GR), glutationa S-transferase (GST) e CK no estriado de ratos. O sulfito ainda aumentou o conteúdo proteico das enzimas superóxido dismutase-1 (SOD1) e catalase (CAT) e diminuiu o conteúdo da heme oxigenase-1 (HO1). Além disso, o sulfito diminuiu a fosforilação da p38 e aumentou a da ERK 1/2, não tendo efeito sobre a fosforilação da JNK. Em relação à sinalização de apoptose, o sulfito aumentou o conteúdo proteico da GSK-3β e da caspase-3 clivada. Por outro lado, o conteúdo da caspase- 9, Bcl-xL e α-sinucleína não foi alterado. O pré-tratamento com o JP4-039 preveniu a diminuição da atividade das enzimas antioxidantes e da CK. O JP4-039 também preveniu as alterações no conteúdo proteico da SOD1, CAT, HO1 e GSK-3β, assim como na fosforilação da p38 e ERK 1/2 e conteúdo da caspase-3. No segundo capítulo desta tese, investigamos os efeitos do tiossulfato, outro metabólito acumulado nas doenças com deficiência da SO, sobre parâmetros de homeostase redox e metabolismo energético em córtex cerebral e cerebelo de ratos neonatos. Para isso, ratos Wistar de 1 dia receberam, através de injeção intracerebroventricular, tiossulfato (0,5 μmol/g) ou PBS (veículo), sendo eutanasiados 30 minutos após a administração. Em córtex, o tiossulfato diminuiu as concentrações de GSH e a atividade das enzimas SOD, GST e CAT. Além disso, o metabólito aumentou a oxidação de DCFH. Em relação aos parâmetros de metabolismo energético, o tiossulfato reduziu a atividade do complexo II da cadeia transportadora de elétrons. Já em cerebelo, o tiossulfato, reduziu a atividade da enzima SOD e aumentou a atividade das enzimas GST e CAT. Além disso, aumentou o conteúdo de sulfidrilas e, assim como em córtex, de oxidação de DCFH. O tiossulfato também causou redução da atividade da enzima CK em cerebelo. Desta forma, pode ser presumido que o acúmulo tanto de sulfito como de tiossulfato tem importante papel na fisiopatologia da deficiência da SO uma vez que altas concentrações destes metabólitos induzem estresse oxidativo e disfunção energética além de, no caso do sulfito, causar morte celular. Ainda, foi visto que o JP4-039 pode ser uma importante alternativa terapêutica para melhorar o prognóstico dos pacientes portadores desse distúrbio.Sulfite oxidase (SO) deficiency is an autosomal recessive condition that can manifest in two forms: isolated SO deficiency and molybdenum cofactor deficiency. Isolated SO deficiency occurs due to mutations in the SUOX gene, responsible for encoding the SO apoenzyme itself, leading to the decrease or absence of this activity, and subsequent degradation of the enzyme. The second form is caused by a deficiency in the activity of any enzyme that participates in the molybdenum cofactor biosynthesis route. Since the SO enzyme is responsible for the oxidation of sulfite to sulfate, both forms are characterized by tissue accumulation of sulfite and thiosulphate. Furthermore, they are clinically characterized by severe neonatal seizures, progressive neurological dysfunction, axial hypotonia, peripheral hypertonicity and psychomotor development delay, which commonly result in premature death. Considering that the pathophysiology of the neurological damage observed in SO deficiency is not fully established, the first chapter of this thesis investigated the in vivo effects of sulfite on antioxidant defenses, creatine kinase (CK) activity, MAPK pathway, and apoptosis markers in the striatum of young rats. The influence of the mitochondria-targeted reactive species scavenger JP4-039 was also evaluated on the possible toxic effects of sulfite. For this, 30-day-old rats received a single intrastriatal dose of sulfite (Na2SO3; 2μmol; test group) or NaCl (2μmol; control group), and were euthanized 30 min after the injection. In other groups, rats received two intraperitoneal injections of JP4-039 (one injection of 3.5μg/g and another of 5μg/g or two injections of 5μg/g), 24 and 2 hours before the administration of sulfite or NaCl. After euthanasia, the striatum was removed and homogenized for analysis. Sulfite decreased the concentrations of reduced glutathione (GSH) and the activities of the enzymes glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), glutathione S-transferase (GST) and CK in rat striatum. Sulfite also increased the protein content of the enzymes superoxide dismutase-1 (SOD1) and catalase (CAT) and decreased the content of heme oxygenase-1 (HO1). Furthermore, sulfite decreased p38 phosphorylation and increased that of ERK 1/2, with no effect on JNK phosphorylation. Regarding apoptosis signaling, sulfite increased the protein content of GSK-3β and cleaved caspase-3. On the other hand, the content of caspase-9, Bcl-xL and α-synuclein was not altered. Pre-treatment with JP4-039 prevented the decrease in the activity of antioxidant enzymes and CK. JP4-039 also prevented changes in SOD1, CAT, HO1 and GSK-3β protein content, as well as p38 and ERK 1/2 phosphorylation and caspase-3 content. In the second chapter of this thesis, we investigated the effects of thiosulphate, another metabolite accumulated in SO deficiency disorders, on parameters of redox homeostasis and energy metabolism in the cerebral cortex and cerebellum of newborn rats. For this, 1-day-old Wistar rats received, through intracerebroventricular injection, thiosulfate (0.5 μmol/g) or vehicle, and were euthanized 30 minutes after administration. In the cortex, thiosulphate decreased GSH concentrations and SOD, GST and CAT activities. Furthermore, it increased the oxidation of DCFH. Regarding the bioenergetics parameters, thiosulphate reduced the activity of the respiratory chain complex II. In the cerebellum, thiosulphate reduced the activity of SOD and increased the activity of GST and CAT. In addition, it increased the content of sulfhydryl groups and the oxidation of DCFH. Thiosulphate also caused a decrease in CK activity. Thus, it can be presumed that the accumulation of both sulfite and thiosulphate plays an important role in the pathophysiology of symptoms observed in SO deficiency, since high concentrations of these metabolites can exacerbate oxidative stress and cause energetic failure and, as seen with sulfite, cause cell death. In addition, we suggest that JP4-039 can be an important therapeutic alternative to improve the prognosis of patients with these disorders

Myelin disruption, neuroinflammation, and oxidative stress induced by sulfite in the striatum of rats are mitigated by the pan-PPAR agonist bezafibrate

Sulfite predominantly accumulates in the brain of patients with isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies. Patients present with severe neurological symptoms and basal ganglia alterations, the pathophysiology of which is not fully established. Therapies are ineffective. To elucidate the pathomechanisms of ISOD and MoCD, we investigated the effects of intrastriatal administration of sulfite on myelin structure, neuroinflammation, and oxidative stress in rat striatum. Sulfite administration decreased FluoromyelinTM and myelin basic protein staining, suggesting myelin abnormalities. Sulfite also increased the staining of NG2, a protein marker of oligodendrocyte progenitor cells. In line with this, sulfite also reduced the viability of MO3.13 cells, which express oligodendroglial markers. Furthermore, sulfite altered the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1), indicating neuroinflammation and redox homeostasis disturbances. Iba1 staining, another marker of neuroinflammation, was also increased by sulfite. These data suggest that myelin changes and neuroinflammation induced by sulfite contribute to the pathophysiology of ISOD and MoCD. Notably, post-treatment with bezafibrate (BEZ), a pan-PPAR agonist, mitigated alterations in myelin markers and Iba1 staining, and IL-1β, IL-6, iNOS and HO-1 expression in the striatum. MO3.13 cell viability decrease was further prevented. Moreover, pretreatment with BEZ also attenuated some effects. These findings show the modulation of PPAR as a potential opportunity for therapeutic intervention in these disorders

Structure and intracellular antioxidant activity of pectic polysaccharide from acerola (Malpighia emarginata)

Malpighia emarginata is a tropical fruit plant, found naturally in the Caribbean islands and South America that produces an edible fruit known as acerola or Barbados Cherry. Its polysaccharides were obtained by aqueous extraction, subjected to a freezing and thawing process and ultrafiltration. A homogeneous fraction (ACWS-01E) was analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analyses. The results showed an arabinan-rich pectic polysaccharide, with 6.1×104g/mol and formed mainly by a high methyl esterified (DM=86%) homogalacturonan and branched arabinan. This latter is anchored in type I rhamnogalacturonan regions. The main chain of arabinan consisted of (1→5)-linked α-Araf, branched only at O-3. The potential ACWS-01E intracellular antioxidant activity against H2O2-induced oxidative stress in murine fibroblast cell line (3T3) was determined by DCFH-DA assay. The treatment with ACWS-01E significantly reduced H2O2-induced cytotoxic effect and the levels of reactive oxygen species (ROS). These findings suggested that ACWS-01E protected and improved NIH 3T3 cell viability from H2O2-induced toxicity by decreasing intracellular levels of ROS.</p

Myelin Disruption, Neuroinflammation, and Oxidative Stress Induced by Sulfite in the Striatum of Rats Are Mitigated by the pan-PPAR agonist Bezafibrate

Sulfite predominantly accumulates in the brain of patients with isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies. Patients present with severe neurological symptoms and basal ganglia alterations, the pathophysiology of which is not fully established. Therapies are ineffective. To elucidate the pathomechanisms of ISOD and MoCD, we investigated the effects of intrastriatal administration of sulfite on myelin structure, neuroinflammation, and oxidative stress in rat striatum. Sulfite administration decreased FluoromyelinTM and myelin basic protein staining, suggesting myelin abnormalities. Sulfite also increased the staining of NG2, a protein marker of oligodendrocyte progenitor cells. In line with this, sulfite also reduced the viability of MO3.13 cells, which express oligodendroglial markers. Furthermore, sulfite altered the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1), indicating neuroinflammation and redox homeostasis disturbances. Iba1 staining, another marker of neuroinflammation, was also increased by sulfite. These data suggest that myelin changes and neuroinflammation induced by sulfite contribute to the pathophysiology of ISOD and MoCD. Notably, post-treatment with bezafibrate (BEZ), a pan-PPAR agonist, mitigated alterations in myelin markers and Iba1 staining, and IL-1β, IL-6, iNOS and HO-1 expression in the striatum. MO3.13 cell viability decrease was further prevented. Moreover, pre-treatment with BEZ also attenuated some effects. These findings show the modulation of PPAR as a potential opportunity for therapeutic intervention in these disorders

Antioxidant system disturbances and mitochondrial dysfunction induced by 3-methyglutaric acid in rat heart are prevented by bezafibrate

Barth syndrome (BTHS) and dilated cardiomyopathy with ataxia syndrome (DCMA) are biochemically characterized by high levels of 3-methylglutaric acid (MGA) in the urine and plasma of affected patients. Although cardiolipin abnormalities have been observed in these disorders, their pathophysiology is not fully established. We evaluated the effects of MGA administration on redox homeostasis and mitochondrial function in heart, as well as on vascular reactivity in aorta of Wistar rats without cardiolipin genetic deficiency. Potential cardioprotective effects of a pretreatment with bezafibrate (BEZ), a pan-PPAR agonist that induces mitochondrial biogenesis, were also determined. Our findings showed that MGA induced lipid peroxidation, altered enzymatic and non-enzymatic antioxidant defenses and reduced respiratory chain function in rat heart. MGA also increased Drp1 and reduced MFN1 levels, suggesting mitochondrial fission induction. Moreover, MGA altered MAPK and Akt signaling pathways, and had a strong tendency to reduce Sirt1 and PGC-1α, indicative of mitochondrial biogenesis impairment. Aorta vascular reactivity was further altered by MGA. Additionally, BEZ mitigated most alterations on antioxidant defenses and mitochondrial quality control proteins provoked by MGA. However, vascular reactivity disturbances were not prevented. It may be presumed that oxidative stress, mitochondrial bioenergetics and control quality disturbances, and vascular reactivity impairment caused by MGA may be involved in the cardiac failure observed in BTHS and DCMA, and that BEZ should be considered as a pharmacological candidate for the treatment of these disorders