A suplementação materna com naringina durante a gestação altera o estado redox e a funcionalidade mitocondrial no encéfalo da prole de ratas Wistar

Ao longo das últimas décadas, um acumulado cada vez maior de evidências vem demonstrando que o consumo regular de polifenois leva a diversos benefícios para saúde em modelos experimentais e em estudos clínicos. Da mesma forma, a suplementação com naringina, um polifenol da classe dos flavonoides, é capaz de induzir neuroproteção e resgatar a neurogênese adulta em diferentes modelos experimentais de doenças do sistema nervoso central, principalmente por reduzir o estresse oxidativo e a disfunção mitocondrial. Tais dados causam um enorme impacto na percepção da população sobre o consumo dos suplementos ricos em polifenois, o que leva a altas taxas de consumo, inclusive entre mulheres gestantes, atingindo níveis de 57%. Não surpreendentemente, diversos trabalhos já começaram a demonstram que o consumo materno de polifenois durante a gestação pode levar a alterações metabólicas e funcionas na prole durante a vida adulta. Dessa forma, visto que a naringina é capaz de regular a sinalização redox e mitocondrial, nosso objetivo com o presente trabalho foi avaliar se a suplementação com a naringina durante a terceira semana de gestação pode induzir alterações redox e mitocondriais no encéfalo da prole durante o desenvolvimento pós-natal. Para isso, nós utilizamos ratas Wistar prenhas que foram divididas em dois grupos: controle e naringina (100 mg/kg/dia) e suplementadas via gavagem do dia gestacional 15 a 21. Após o nascimento a prole foi eutanasiada nos dias pós-natais 1, 7 e 21 e o cerebelo, hipocampo, córtex pré-frontal e corpo estriado foram coletados para as análises. Além disso também realizamos a coleta de sangue para perfil bioquímico e análise do desenvolvimento neuromotor nas duas primeiras semanas de vida da prole. Nossos resultados mostram que a suplementação materna com naringina levou ao aumento do colesterol total e HDL aos 21 de vida da prole, sem alterar os outros parâmetros metabólicos. Além disso, também observamos diversas alterações redox de forma região e sexo-específica no encéfalo da prole que se concentraram principalmente no período perinatal. No entanto, tais alterações não persistiram até o desmame, provavelmente pela redução da ativação do NRF2 que pode ter sido induzida no período da suplementação. Já as alterações mitocondriais encontradas no cerebelo demonstraram um caráter sexodependente durante o período perinatal e persistiram até o desmame. Aos 21 dias de vida, a prole nascida de ratas suplementadas com naringina apresentou maior funcionalidade mitocondrial, produção de superóxido e redução da proteína DRP1, indicando redução da fissão mitocondrial. Tais alterações foram acompanhadas pelo aumento da expressão da sirtuína 3 e redução da ativação da proteína AKT sem a indução de dano oxidativo às proteínas. Nossos achados indicam que a suplementação materna com naringina induziu uma adaptação metabólica no cerebelo da prole, onde ela foi capaz de estimular de forma persistente a respiração e metabolismo mitocondrial, levando a uma ativação da sirtuína 3 e uma alteração morfológica mitocondrial frente a tal adaptação. Assim, é importante considerar cuidadosamente o consumo de suplementos contendo altas doses de polifenois durante a gestação, visto que nossos dados demonstram que eles podem modular a sinalização, dinâmica e funcionalidade mitocondrial, e consequentemente interferir durante o processo de neurogênese e neurodesenvolvimento fetal.During the last decades, an increasing body of evidence has been demonstrating that regular polyphenol consumption may induce several health benefits in both experimental and clinical studies. Similarly, naringin supplementation is also able to induce neuroprotection and improve adult neurogenesis in different models of central nervous system diseases, mainly by reducing oxidative stress and mitochondrial dysfunction. Such a body of evidence cause a strong impact on the population’s perception of the consumption of polyphenol-rich supplements, which consequently leads to high rates of polyphenol supplement consumption, even among specific groups, such as pregnant women, who have consumption rates of polyphenol-rich supplements of up to 57%. Not surprisingly, several experimental and clinical studies have stater to report that high polyphenol intake during gestation may induce metabolic and functional alterations in the offspring even during postnatal development. Considering the above information, the main objective of the present work was to evaluate if maternal naringin supplementation during the third week of pregnancy could induce redox and mitochondrial alterations in the offspring’s brain during their postnatal development. We used pregnant Wistar rats that were divided into two groups: control and naringin (100 mg/kg/day) and were orally supplemented from gestational day 15 until gestational day 21. On postnatal days 1, 7, and 21 the offspring were euthanized, and the cerebellum, hippocampus, prefrontal cortex, and striatum were dissected. Moreover, we also collected the offspring and mother’s trunk blood for metabolic profiling and evaluated the physical and neuromotor offspring’s development during the first two weeks of life. Our results showed that the maternal naringin supplementation increased the offspring’s total cholesterol and HDL levels but did not affect the other blood parameters analyzed. In addition, we also observed several redox alterations in a sex and region-specific manner in the offspring’s brain, that were mainly found during the perinatal period. However, such alterations did not persist up to weaning age, probably because of the reduced NRF2 activation that was once triggered by naringin during the third week of gestation. On the other hand, the mitochondrial alterations found in the pups’ cerebellum during the perinatal period were mostly sex-dependent and persisted up to postnatal day 21, in which they affected both sexes. At weaning age, the offspring born from supplemented rats showed increased mitochondrial functionality, superoxide production e reduced DRP1 protein content, suggesting reduced mitochondrial fission. Such alterations were accompanied by a 2-fold increase in sirtuin 3 protein content, reduced AKT activation, and no protein oxidative damage. Therefore, our findings suggest that maternal naringin supplementation induced a metabolic adaptation in the offspring’s cerebellum probably by persistently stimulating the mitochondrial respiration and metabolism during the prenatal period. Such a stimulation consequently led to the sirtuin 3 activation and an alteration in the mitochondrial morphology to meet the metabolic adaptation. In conclusion, the data highlight the importance of carefully considering whether high polyphenol supplementation should be advised or not during gestation since our findings showed that naringin was capable to modulate the redox and mitochondrial signaling, which may consequently interfere during fetal neurogenesis and neurodevelopment

Naringin supplementation during pregnancy induces sex and region-specific alterations in the offspring’s brain redox status

Research has shown the beneficial effects of naringin supplementation to adult rodents, which can ameliorate oxidative stress in disease models. However, evidence has demonstrated that polyphenol supplementation induced detrimental effects when consumed during sensitive periods of development, such as pregnancy. Therefore, we investigated the effect of maternal naringin supplementation during pregnancy on the offspring’s cerebral redox status. Pregnant Wistar rats were divided into control and naringin groups and supplemented from gestational day 15 to gestational day 21. On postnatal days 1, 7, and 21, offspring were euthanized, and the prefrontal cortex, hippocampus, striatum, and cerebellum dissected. On postnatal day 1, maternal naringin supplementation positively modulated the pups’ brain redox status. On postnatal day 7, a pro-oxidative milieu was observed in the offspring’s striatum and cerebellum in a sex-dependent manner, even though the prefrontal cortex and hippocampus were not negatively affected. Besides, the alterations observed on postnatal day 7 did not persist up to weaning. Our findings demonstrated that the effect induced by naringin supplementation in the brain redox status differed according to the period of development in which naringin was consumed since the beneficial effects usually found in the adult rodents became detrimental when the supplementation was applied during pregnancy

Physical Exercise During Pregnancy Prevents Cognitive Impairment Induced by Amyloid-ß in Adult Offspring Rats

Alzheimer’s disease (AD) is the main aging-associated neurodegenerative disorder and is characterized by mitochondrial dysfunction, oxidative stress, synaptic failure, and cognitive decline. It has been a challenge to find disease course-modifying treatments. However, several studies demonstrated that regular physical activity and exercise are capable of promoting brain health by improving the cognitive function. Maternal lifestyle, including regular exercise during pregnancy, has also been shown to influence fetal development and disease susceptibility in adulthood through fetal metabolism programming. Here, we investigated the potential neuroprotective role of regular maternal swimming, before and during pregnancy, against amyloid-β neurotoxicity in the adult offspring. Behavioral and neurochemical analyses were performed 14 days after male offspring received a single, bilateral, intracerebroventricular (icv) injection of amyloid-β oligomers (AβOs). AβOs-injected rats of the sedentary maternal group exhibited learning and memory deficits, along with reduced synaptophysin, brain-derived neurotrophic factor (BDNF) levels, and alterations of mitochondrial function. Strikingly, the offspring of the sedentary maternal group had AβOs-induced behavioral alterations that were prevented by maternal exercise. This effect was accompanied by preventing the alteration of synaptophysin levels in the offspring of exercised dams. Additionally, offspring of the maternal exercise group exhibited an augmentation of functional mitochondria, as indicated by increases in mitochondrial mass and membrane potential, α-ketoglutarate dehydrogenase, and cytochrome c oxidase enzymes activities. Moreover, maternal exercise during pregnancy induced long-lasting modulation of fusion and fission proteins, Mfn1 and Drp1, respectively. Overall, our data demonstrates a potential protective effect of exercise during pregnancy against AβOs-induced neurotoxicity in the adult offspring brain, by mitigating the neurodegenerative process triggered by Alzheimer-associated AβOs through programming the brain metabolism.This study was supported by the Pró-Reitoria de Pesquisa/Universidade Federal do Rio Grande do Sul (PROPESQ/UFRGS). CPK is a PhD Postgraduate student in Biological Sciences – Biochemistry receiving grants from the Brazilian agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). CM received grants from CNPq (Universal 442406/2014-2 and INCT 465671/2014-4)

A suplementação pré-natal com naringina altera a homeostase redox e função mitocondrial cerebral da prole de ratas Wistar

Diversos trabalhos vêm demonstrando que a suplementação com naringina é capaz de promover a neuroproteção em modelos de doenças neurodegenerativas induzidos em animais, principalmente através da regulação da homeostase redox e função mitocondrial. Embora a naringina possa ser considerada como uma possível abordagem terapêutica nesses modelos, ainda se desconhecem os efeitos que essa suplementação pode ter quando realizada durante a gestação no desenvolvimento fetal. Dessa forma, nós buscamos avaliar o efeito da suplementação com naringina durante a gestação sobre a homeostase redox e a função mitocondrial no encéfalo de filhotes fêmeas, além de investigar parâmetros de desenvolvimento pós-natal e memória nos filhotes machos nascidos de ratas que foram suplementadas. Assim, ratas Wistar prenhas foram divididas em dois grupos: (1) controle e (2) naringina, que foi administrada na dose de 100 mg/kg/dia por via oral durante toda a prenhez. Após o nascimento, as filhotes fêmeas foram eutanasiados nos dias pós-natais 1, 7 e 21, e córtex pré-frontal, hipocampo, corpo estriado e cerebelo foram coletados para análises bioquímicas. Os filhotes machos tiveram seu desenvolvimento pós-natal avaliado durante as duas primeiras semanas depois do nascimento, entre os dias pós-natais 2 e 15, além de serem submetidos a testes comportamentais no dia pós-natal 21. Nossos resultados demonstraram que a suplementação com naringina durante a gestação alterou a homeostase redox e a função mitocondrial no encéfalo das fêmeas de uma forma específica em cada estrutura e que essas alterações também foram dependentes da idade avaliada, já que a maior intensidade de efeitos foi observada nos dias pós-natais 1 e 7, e reduziram-se progressivamente até o dia pós-natal 21, exceto no cerebelo, que manteve um número elevado de alterações até essa idade, demonstrando ser mais susceptível aos efeitos do consumo materno de naringina durante a gestação. Os filhotes machos não apresentaram alterações significativas tanto em seu desenvolvimento durante as duas primeiras semanas após o nascimento quanto no desempenho nos testes comportamentais para avaliação da função mnemônica no dia pós-natal 21. Dessa forma, concluímos que a suplementação com naringina durante a gestação pode ser prejudicial ao desenvolvimento do sistema nervoso central em relação à homeostase redox e à função mitocondrial, especialmente no cerebelo, visto que essa suplementação foi capaz de desencadear diversas alterações bioquímicas. Se nossos dados puderem ser extrapolados para a clínica, sugerimos atenção ao consumo e suplementação com polifenóis durante o período gestacional, considerando os possíveis efeitos deletérios sobre o cérebro em desenvolvimento.Several studies have been demonstrating that naringin supplementation promotes neuroprotection when used in animal models of neurodegenerative diseases, especially by regulating redox homeostasis and mitochondrial function. However, despite naringin use as a potential therapeutic approach in those models, there is no data concerning the effects of naringin supplementation during pregnancy on fetal development. Thereby, we sought to evaluate the effects of maternal naringin supplementation during pregnancy on redox homeostasis and mitochondrial function in female pups’ brain, as well as on postnatal development and memory of male pups. Thus, pregnant Wistar rats were divided into two groups: (1) control and (2) naringin, which was supplemented by oral gavage in a dose of 100 mg/kg/day during entire pregnancy. After birth, female pups were euthanized on postnatal days 1, 7 and 21, and prefrontal cortex, hippocampus, striatum, and cerebellum were dissected for biochemical essays. Male pups were used to asses postnatal development during the first two weeks after birth, between postnatal days 2 and 15, and were submitted to behavioral test on postnatal day 21. Our results demonstrated that naringin supplementation during pregnancy altered redox homeostasis and mitochondrial function in brain of female pups in a structure specific manner, and that these alterations were also time-dependent, since most alterations were found on postnatal days 1 and 7, and progressively declined until postnatal day 21, except the cerebellum, which demonstrated an increased number of alterations until the last age, suggesting a higher susceptibility of this brain structure to the programming effects triggered by maternal naringin supplementation during gestation. Moreover, male pups did not demonstrate any significant alteration during postnatal development on the first two weeks after birth or on behavioral tests on postnatal day 21. Thereby, maternal naringin supplementation during pregnancy might be detrimental to the central nervous system development when considering redox homeostasis and mitochondrial function, especially on cerebellum, since naringin supplementation triggered several biochemical alterations. Whether our data could be considered for clinical purposes, we suggest attention to polyphenol consumption and supplementation during pregnancy, considering the high impact on brain metabolism during development

A suplementação pré-natal com naringina altera a homeostase redox e função mitocondrial cerebral da prole de ratas Wistar

Diversos trabalhos vêm demonstrando que a suplementação com naringina é capaz de promover a neuroproteção em modelos de doenças neurodegenerativas induzidos em animais, principalmente através da regulação da homeostase redox e função mitocondrial. Embora a naringina possa ser considerada como uma possível abordagem terapêutica nesses modelos, ainda se desconhecem os efeitos que essa suplementação pode ter quando realizada durante a gestação no desenvolvimento fetal. Dessa forma, nós buscamos avaliar o efeito da suplementação com naringina durante a gestação sobre a homeostase redox e a função mitocondrial no encéfalo de filhotes fêmeas, além de investigar parâmetros de desenvolvimento pós-natal e memória nos filhotes machos nascidos de ratas que foram suplementadas. Assim, ratas Wistar prenhas foram divididas em dois grupos: (1) controle e (2) naringina, que foi administrada na dose de 100 mg/kg/dia por via oral durante toda a prenhez. Após o nascimento, as filhotes fêmeas foram eutanasiados nos dias pós-natais 1, 7 e 21, e córtex pré-frontal, hipocampo, corpo estriado e cerebelo foram coletados para análises bioquímicas. Os filhotes machos tiveram seu desenvolvimento pós-natal avaliado durante as duas primeiras semanas depois do nascimento, entre os dias pós-natais 2 e 15, além de serem submetidos a testes comportamentais no dia pós-natal 21. Nossos resultados demonstraram que a suplementação com naringina durante a gestação alterou a homeostase redox e a função mitocondrial no encéfalo das fêmeas de uma forma específica em cada estrutura e que essas alterações também foram dependentes da idade avaliada, já que a maior intensidade de efeitos foi observada nos dias pós-natais 1 e 7, e reduziram-se progressivamente até o dia pós-natal 21, exceto no cerebelo, que manteve um número elevado de alterações até essa idade, demonstrando ser mais susceptível aos efeitos do consumo materno de naringina durante a gestação. Os filhotes machos não apresentaram alterações significativas tanto em seu desenvolvimento durante as duas primeiras semanas após o nascimento quanto no desempenho nos testes comportamentais para avaliação da função mnemônica no dia pós-natal 21. Dessa forma, concluímos que a suplementação com naringina durante a gestação pode ser prejudicial ao desenvolvimento do sistema nervoso central em relação à homeostase redox e à função mitocondrial, especialmente no cerebelo, visto que essa suplementação foi capaz de desencadear diversas alterações bioquímicas. Se nossos dados puderem ser extrapolados para a clínica, sugerimos atenção ao consumo e suplementação com polifenóis durante o período gestacional, considerando os possíveis efeitos deletérios sobre o cérebro em desenvolvimento.Several studies have been demonstrating that naringin supplementation promotes neuroprotection when used in animal models of neurodegenerative diseases, especially by regulating redox homeostasis and mitochondrial function. However, despite naringin use as a potential therapeutic approach in those models, there is no data concerning the effects of naringin supplementation during pregnancy on fetal development. Thereby, we sought to evaluate the effects of maternal naringin supplementation during pregnancy on redox homeostasis and mitochondrial function in female pups’ brain, as well as on postnatal development and memory of male pups. Thus, pregnant Wistar rats were divided into two groups: (1) control and (2) naringin, which was supplemented by oral gavage in a dose of 100 mg/kg/day during entire pregnancy. After birth, female pups were euthanized on postnatal days 1, 7 and 21, and prefrontal cortex, hippocampus, striatum, and cerebellum were dissected for biochemical essays. Male pups were used to asses postnatal development during the first two weeks after birth, between postnatal days 2 and 15, and were submitted to behavioral test on postnatal day 21. Our results demonstrated that naringin supplementation during pregnancy altered redox homeostasis and mitochondrial function in brain of female pups in a structure specific manner, and that these alterations were also time-dependent, since most alterations were found on postnatal days 1 and 7, and progressively declined until postnatal day 21, except the cerebellum, which demonstrated an increased number of alterations until the last age, suggesting a higher susceptibility of this brain structure to the programming effects triggered by maternal naringin supplementation during gestation. Moreover, male pups did not demonstrate any significant alteration during postnatal development on the first two weeks after birth or on behavioral tests on postnatal day 21. Thereby, maternal naringin supplementation during pregnancy might be detrimental to the central nervous system development when considering redox homeostasis and mitochondrial function, especially on cerebellum, since naringin supplementation triggered several biochemical alterations. Whether our data could be considered for clinical purposes, we suggest attention to polyphenol consumption and supplementation during pregnancy, considering the high impact on brain metabolism during development

Maternal exercise during pregnancy modulates mitochondrial function and redox status in a sex-dependent way in adult offspring’s skeletal muscle

Maternal exercise has shown beneficial effects on mother and child. Literature confirm progeny’s cognition improvement, and upregulation in neurotrophins, antioxidant network, and DNA repair system. Considering that there is a lack of information demonstrating the impact of maternal exercise on offspring’s skeletal muscle, we aimed to investigate the mitochondrial and redox effects elicited by maternal swimming. Adult female Wistar rats were divided into three groups: control sedentary, free swimming, and swimming with overload (2% of the body weight). Exercised groups were submitted weekly to five swimming sessions (30 min/day), starting 1 week prior to the mating and lasting to the delivery. Gastrocnemius and soleus muscle from 60-day-old offspring were analyzed. Our results clearly showed a sex-dependent effect. Male soleus showed increased mitochondrial functionality in the overload group. Female muscle from the overload group adapted deeply. Considering the redox status, the female offspring delivered to overload exercised dams presented reduced oxidants levels and protein damage, allied to downregulated antioxidant defenses. We also observed an increase in the mitochondrial function in the gastrocnemius muscle of the female offspring born from overload exercised dams. Soleus from female delivered to the overload exercise group presented reduced mitochondrial activity, as well as reduced reactive species, protein carbonyls, and antioxidant network, when compared to the male. In conclusion, maternal exercise altered the redox status and mitochondrial function in the offspring’s skeletal muscle in a sex-dependent way. The clinical implication was not investigated; however, the sexual dimorphism in response to maternal exercise might impact exercise resilience in adulthood

Naringin Supplementation during Pregnancy Induces Sex and Region-Specific Alterations in the Offspring’s Brain Redox Status

Research has shown the beneficial effects of naringin supplementation to adult rodents, which can ameliorate oxidative stress in disease models. However, evidence has demonstrated that polyphenol supplementation induced detrimental effects when consumed during sensitive periods of development, such as pregnancy. Therefore, we investigated the effect of maternal naringin supplementation during pregnancy on the offspring’s cerebral redox status. Pregnant Wistar rats were divided into control and naringin groups and supplemented from gestational day 15 to gestational day 21. On postnatal days 1, 7, and 21, offspring were euthanized, and the prefrontal cortex, hippocampus, striatum, and cerebellum dissected. On postnatal day 1, maternal naringin supplementation positively modulated the pups’ brain redox status. On postnatal day 7, a pro-oxidative milieu was observed in the offspring’s striatum and cerebellum in a sex-dependent manner, even though the prefrontal cortex and hippocampus were not negatively affected. Besides, the alterations observed on postnatal day 7 did not persist up to weaning. Our findings demonstrated that the effect induced by naringin supplementation in the brain redox status differed according to the period of development in which naringin was consumed since the beneficial effects usually found in the adult rodents became detrimental when the supplementation was applied during pregnancy

Behavioral benefits of maternal swimming are counteracted by neonatal hypoxia-ischemia in the offspring

Hypoxia-ischemia (HI) represents one of the most common causes of neonatal encephalopathy. The central nervous system injury comprises several mechanisms, including inflammatory, excitotoxicity, and redox homeostasis unbalance leading to cell death and cognitive impairment. Exercise during pregnancy is a potential therapeutic tool due to benefits offered to mother and fetus. Swimming during pregnancy elicits a strong metabolic programming in the offspring’s brain, evidenced by increased antioxidant enzymes, mitochondrial biogenesis, and neurogenesis. This article aims to evaluate whether the benefits of maternal exercise are able to prevent behavioral brain injury caused by neonatal HI. Female adult Wistar rats swam before and during pregnancy (30 min/day, 5 days/week, 4 weeks). At 7th day after birth, the offspring was submitted to HI protocol and, in adulthood (60th day), it performed the behavioral tests. It was observed an increase in motor activity in the open field test in HI-rats, which was not prevented by maternal exercise. The rats subjected to maternal swimming presented an improved long-term memory in the object recognition task, which was totally reversed by neonatal HI encephalopathy. BDNF brain levels were not altered; suggesting that HI or maternal exercise effects were BDNF-independent. In summary, our data suggest a beneficial long-term effect of maternal swimming, despite not being robust enough to protect from HI injury

Protective effect of maternal exercise against amyloid-β neurotoxicity in the male rat offspring’s cerebellum

The Developmental Origins of Health and Disease (DOHaD) states that intrauterine maternal environment influences postnatal life by programming offspring’s metabolism. Intrauterine milieu induced by exercise during pregnancy promotes long-lasting benefits to the offspring’s health and seems to offer some resistance against chronic diseases in adult life. Alzheimer’s disease is a public health concern with limited treatment options. In the present study, we assessed the potential of maternal exercise during pregnancy in long-term programming of young adult male rat offspring’s cerebellar metabolism in conferring neuroprotection against amyloid-β (Aβ) neurotoxicity. Female Wistar rats were submitted to a swimming protocol 1 week prior mating and throughout pregnancy (five sessions/a week lasting 30 min). Aβ oligomers were infused bilaterally in the brain ventricles of 60-day-old male offspring. Fourteen days after surgery, we measured parameters related to redox state, mitochondrial function, and the immunocontent of proteins related to synaptic function. We found that maternal exercise during pregnancy attenuated several parameters in the offspring’s male rat cerebellum, such as the reactive species rise, the increase of inducible nitric oxide synthase immunocontent and tau phosphorylation induced by Aβ oligomers, increased mitochondrial fission indicated by dynamin-related protein 1 (DRP1), and protein oxidation identified by carbonylation. Strikingly, we find that maternal exercise promotes changes in the rat offspring’s cerebellum that are still evident in young adult life. These favorable neurochemical changes in offspring’s cerebellum induced by maternal exercise may contribute to a protective phenotype against Aβ-induced neurotoxicity in young adult male rat offspring.This study was supported by the Pró-Reitoria de Pesquisa/ Universidade Federal do Rio Grande do Sul (PROPESQ/UFRGS). Financial support. CPK is a PhD postgraduate student in Biological Sciences –Biochemistry receiving grants from the Brazilian agency Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq). CM received grants from CNPq [Universal 442406/2014-2 and INCT 465671/2014-4].Peer reviewe