Oridonin attenuates the progression of atherosclerosis by inhibiting NLRP3 and activating Nrf2 in apolipoprotein E-deficient mice
Lei WangXiaoqi ZhaoJiawen DingYutong LiuHan LiuLei ZhengHongting ZhaoZichen SunKuanyu LiJing CaiTong Qiao
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Abstract Oridonin, a well-known traditional Chinese herbal medicinal product isolated from Isodon rubescens (Hemsl.) H.Hara, has many potential properties, including anti-inflammatory and antioxidant activities. However, there is no evidence whether oridonin have a protective effect on atherosclerosis. This study focused on the effects of oridonin on oxidative stress and inflammation generated from atherosclerosis. The therapeutic effect on atherosclerosis was evaluated by intraperitoneal injection of oridonin in a high-fat fed ApoE −/− mouse model. We isolated mouse peritoneal macrophages and detected the effect of oridonin on oxidized low-density lipoprotein-induced lipid deposition. Oil red O staining, Masson's staining, Dihydroethidium (DHE) fluorescence staining, Immunohistochemical staining, western blotting analysis, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR were used to evaluate the effect on atherosclerosis and explore the mechanisms. Oridonin treatment significantly alleviated the progression of atherosclerosis, reduced macrophage infiltration and stabilized plaques. Oridonin could significantly inhibit inflammation associated with NLRP3 activation. Oridonin significantly reduced oxidative stress by blocking Nrf2 ubiquitination and degradation. We also found that oridonin could prevent the formation of foam cells by increasing lipid efflux protein and reducing lipid uptake protein in macrophages. Oridonin has a protective effect on atherosclerosis in ApoE −/− mice, which may be related to the inhibition of NLRP3 and the stabilization of Nrf2. Therefore, oridonin may be a potential therapeutic agent for atherosclerosis.Keywords:
Oil Red O
Apolipoprotein E
Intraperitoneal injection
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Amyloid (mycology)
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The strongest risk-determinant for the development of sporadic Alzheimer's disease (AD) is the polymorphic APOE gene. In humans APOE has three variants, APOEε2, APOEε3 and APOEε4 whereof the latter increases the risk of disease 4–15 fold in a dose-dependent manner whilst APOEε2 appears to be protective. The biological mechanisms underlying the modified risk of disease in APOEε2 and APOEε4 carriers are not known. We previously showed that APOEε4-carriers exhibit a prominent plasma apolipoprotein E (apoE) deficiency caused by a specific reduction of the apoE4 isoform. This apoE deficiency was not observed in cerebrospinal fluid. In cognitively intact APOEε3/ε4 carriers an increased relative ratio of plasma apoE4 to apoE3 correlated to glucose hypometabolism and gray matter volume reductions in brain areas most often affected in AD. Hence, we speculate that peripheral apoE levels are linked to processes driving the risk of developing AD brain pathology. In order to determine the cause of the observed phenotype of plasma apoE deficiency in APOEε4-carriers we aimed to investigate the expression of different APOE alleles in liver biopsies from individuals with an APOEε3/ε4 versus an APOEε2/ε3 genotype. Liver biopsies from liver explants were received from n=3 APOEε3/ε4 and n=3 APOEε2/ε3 carriers who had undergone liver transplantation at the Karolinska University Hospital in Sweden. Total RNA (≥80% in DV200 score) was isolated according to routine laboratory methods and used for RNA sequencing employing the high-throughput Illumina platform. Paired-end sequencing reads were aligned to the human reference genome (hg19), using TopHat, and gene counts for expression determined using HTseq-count. Differential expression between the genotype groups was calculated using DEseq2. Preliminary analyses revealed differential expression of n=624 genes between individuals with an APOEε3/ε4 versus an APOEε2/ε3 genotype. In total n=624 genes are differentially expressed in livers from APOEε3/ε4 versus APOEε2/ε3 carriers. Further analyses will reveal the identity of the differentially expressed genes and whether there is a specific difference in the expression of APOE alleles that could explain the observed APOEε4 related plasma apoE deficiency
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Apolipoprotein E (APOE)genotype is the strongest genetic risk factor for late-onset Alzheimer'sdisease (LOAD). APOE protein levels can be measured in the cerebrospinal fluid (CSF) and plasma. Previous studies have failed to detect an association between APOE genotype and CSF levels of APOE. In this project, we tested whether APOE genotype or other variants in the APOE-TOMM40 region are associated with either CSF or plasma APOE levels. APOE protein levels in the cerebrospinal fluid and plasma were quantified using the Luminex xMAP Multiplexing Technology in 349 samples. Expression studies were carried out using cDNA obtained from the parietal lobes of 82 AD cases and 39 cognitively normal individuals (CDR = 0). The SNPs that determine APOE genotype and 20 other SNPs in the APOE-TOMM40 region were genotyped by TaqMan or KASpar technology. In our dataset, CSF and plasma APOE levels are not significantly correlated (R2=0.10;p = 0.08), however APOE genotype is strongly associated with both CSF (R2=0.14; p = 2.4x10−11) and plasma APOE levels (R2=0.26; p = 2.4x10−24). No association was found with any other genotyped SNP in the APOE-TOMM40 region when APOE genotype was included as covariate in the model. No association between APOE genotype and APOE mRNA expression was found (R2=0.05; p = 0.28). We confirmed that APOE genotype is associated with plasma APOE protein levels and failed to observe evidence for any other variation in this region influencing plasma APOE. In contrast to previous results we also observed a strong association between APOE genotype and APOE protein levels in CSF. This may be due to sample size or differences in the ELISA used to measure APOE levels. We did not find an association with APOE gene expression levels. The differences in APOE protein levels in CSF may be due to differences in APOE clearance or degradation resulting from differences in affinity of the APOE isoforms for its receptor.
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Apolipoprotein E (apoE) is associated with lipoproteins in the cerebrospinal fluid (CSF). APOE4 increases and APOE2 decreases the risk for Alzheimer disease (AD) compared to the risk associated with APOE3 Because apoE4 is less efficient at cholesterol efflux than apoE2 or apoE3 in vitro, we hypothesized that APOE genotype may affect apoE particle size in vivo and that these size differences may be related to AD risk. We used nondenaturing gel electrophoresis to test for differences in the size of apoE complexes in human CSF samples of various APOE genotypes and created profiles of each sample to compare the patterns of apoE distribution. For middle-aged adults with no dementia, APOE 2.3 individuals had significantly larger apoE complexes than APOE 3.3 subjects, who had significantly larger apoE complexes than APOE 3.4 and APOE 4.4 individuals. Similarly, in an independent cohort of older adults, CSF apoE complexes of APOE4-positive individuals were smaller than those of the APOE4-negative individuals. Compared to individuals with no dementia, those with the mildest stages of dementia had similar sized CSF apoE complexes. These results identify a novel phenotypic difference in the size of CSF apoE complexes in middle age that correlate with the risk of AD later in life.
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Н. П. Боровкова1, В. А. Шереметьева2, А.
Н. Евсюков3, В. А. Спицын1 ... 1 Медико-генетический научный центр РАМН,
Москва ... 2 Кафедра антропологии биологического факультета
МГУ, Москва ... 3 Институт общей генетики РАН, Москва ... Представлен генетико-антропологический анализ полиморфизма
аполипопротеина E (APOE) среди мирового народонаселения. Пропорции
трех аллелей APOE варьируют в популяциях человека в следующих
весьма широких пределах: APOE*2 - 0 - 37%, APOE*3 - 36 - 98% и
APOE*4 - 0 - 49%. По результатам отечественных и зарубежных
исследований сформирована база данных о частотах аллелей APOE в 372
популяциях мира. Установлены этноантропологические и экологические
особенности панойкуменного распределения полиморфизма APOE.
Рассмотрена гипотеза об адаптивной ценности аллеля APOE*4 в
популяциях, у представителей которых наблюдается сниженная
эффективность синтеза витамина D в организме в результате слабой
инсоляции или в случае выраженной пигментации кожи.
Темнопигментированные группы населения, а также популяции,
локализованные в северных регионах Евразии, характеризуются
увеличением пропорции аллеля APOE*4. Полученные наблюдения
согласуются с гипотезой о преимуществе обладателей этого аллеля в
популяциях, восполняющих недостаточный синтез витамина D в коже за
счет его поглощения с пищей. ... Ключевые слова: аполипопротеин E, полиморфизм, мировое
распределение аллелей APOE, адаптивная значимость
APOE*4 ... УДК 572.2 ... Введение ... Аполипопротеины принадлежат к белковым фрагментам
липопротеиновых частиц, которые функционируют как переносчики
липидов в крови. Этим белкам свойственен генетически
детерминированный структурный полиморфизм и этнические различия в
распределении частот аллелей [Tsunoda et al., 2002]. ... Аполипопротеин E (apoE) является белком с молекулярной массой
34.2 кДа (299 аминокислотных остатков) и кодируется геном
APOE (OMIM 107741), расположенным на хромосоме 19 в локусе
19q13.2. Полиморфизм гена APOE (*2/*3/*4) в популяциях
человека характеризуется 6 генотипами: три гомозиготы (*2/*2,
*3/*3, *4/*4) и три гетерозиготы (*2/*3, *2/*4, *3/*4) [Siest et
al., 1995]. ... Полиморфизм *2/*3/*4 является наиболее изученным для гена
APOE и идентифицирован во многих популяциях мира. Он
оказывает дифференциальное влияние на метаболизм липопротеинов.
Уровень холестерина в организме последовательно повышается в
зависимости от аллельной принадлежности, возрастая в ряду *2 <
*3 < *4, и этот факт является закономерным для разных популяций
[цит. по: Виноградова, 2006]. Кроме метаболизма липидов,
липопротеины имеют также другие важные функции, включая транспорт
гормонов, протеолитическую активность, регуляцию фактора
коагуляции, участие в механизмах защиты и регенерации [Siest et
al., 1995]. ... Исследования последнего десятилетия установили роль
APOE*4 как фактора риска для возникновения болезни
Альцгеймера (БА). У европейцев и евроамериканцев аллель
APOE*4 ассоциирован с повышенным риском развития БА. При БА
гетерозиготы по *4 встречаются в 1.5 - 3 раза чаще, а гомозиготы -
в 2.5 - 10 раз чаще, чем в популяции в целом [цит. по: Боринская и
др., 2007]. Риск возникновения БА у гетерозигот по *4 аллелю выше в
2.7 раза, а у гомозигот - в 9.3 раза [Kuusisto et al., 1994].
Однако у бушменов и ряда других народов Африки этот аллель не
является фактором риска, что предположительно связано с
генетиче- ... ским своеобразием африканцев, живущих к югу от Сахары. Возможна
также связь со специфическими условиями жизни. У афроамериканцев
имеет место ассоциация *4 аллеля с БА, но она выражена в меньшей
степени, чем у субъектов европейского происхождения, что также
свидетельствует о возможном влиянии генетического фона и факторов
среды [цит. по: Боринская и др., 2007]. Таким образом, наличие
аллеля APOE*4 нельзя назвать ни необходимым, ни достаточным
для развития БА. Вероятно, что в этом сложном процессе
задействованы также другие генетические системы и условия
окружающей среды [Kuusisto et al., 1997]. ... Присутствие аллеля APOE*4 рассматривается как фактор
риска развития сердечно-сосудистых заболеваний. Y. Song с коллегами
[2004] сопоставили данные 48 исследований, имеющих отношение к
связи сердечно-сосудистых заболеваний с полиморфизмом гена
APOE. Этот мета-анализ был проведен на базе 15 492 больных
ишемической болезнью сердца (ИБС) и 32 965 лиц контрольной группы.
Было установлено, что риск развития ИБС среди носителей аллеля
APOE*4 выше на 42% по сравнению с обладателями генотипа
APOE*3/*3. Существенных связей между аллелем *2 и риском
развития ИБС обнаружено не было. ... Полиморфизм гена APOE является в своем роде уникальным
явлением для Homo sapiens, поскольку у других млекопитающих,
включая приматов, обнаружена лишь одна форма apoE, которая более
всего схожа с человеческой изоформой apoE*4 [цит. по: Finch and
Sapolsky, 1999]. Аллели APOE*3 и *2 предположительно
возникли в результате мутационных событий, приведших к
последовательным однонуклеотидным заменам.
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Abstract Background Restrictions on mouse models have significantly impacted research towards understanding the most common genotype contributing to dementia in the human population – APOE ε3/ε4 . To address this, as part of MODEL-AD, we created new versions of humanized APOE ε4 and APOE ε3 mice on a C57BL/6J background that allow for unrestricted distribution and breeding. Methods To determine similarities and differences between APOE ε3/ε4 and APOE ε4/ε4 risk genotypes, we analyzed peripheral lipid concentrations as well as performed unbiased transcriptional profiling of the cortex at two and four months of age, comparing APOE ε3/ε4 and APOE ε4/ε4 to the reference APOE ε3/ε3 . To further compare APOE genotypes, cohorts of APOE ε3/ε3 , APOE ε3/ε4 , and APOE ε4/ε4 mice were exercised by voluntary running from 1 month to 4 months of age. Results Cholesterol composition was significantly influenced by APOE genotype as early as 2 months, while triglycerides were affected by APOE genotype at 4 months. Importantly, RNA-sequencing of the cortex followed by linear modeling or weighted gene co-expression network analysis (WGCNA) revealed that the APOE ε3/ε4 genotype showed unique transcriptomic signatures to that of APOE ε4/ε4 . Functional enrichment of the APOE ε3/ε4 , but not APOE ε3/ε4 genotype, revealed sulfur and heparin binding as significant terms at 2 months, and extracellular matrix and blood coagulation at 4 months. Further, cell specific contributions of significant genes identified endothelial cells as overrepresented in the APOE ε3/ε4 but not APOE ε4/ε4 genotype. WGCNA analysis confirmed findings from linear modeling but also predicted that running at a young age affects myelination and gliogenesis across APOE genotypes. Conclusions In summary, APOE ε3/ε4 genotype-specific effects were observed in cortical transcriptional profiles, suggesting therapies aimed at modifying APOE biology to treat dementias may need to be targeted to specific APOE genotypes.
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The ε4 allele of apolipoprotein E (APOE) is currently the major genetic risk factor identified for Alzheimer's disease (AD). Previous in vivo data from our laboratory has demonstrated that amyloid-β (Aβ) is rapidly removed from the plasma by the live
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Generation of reactive oxygen species (ROS) is a normal process. Under physiological conditions, these deleterious species are mostly removed by the cellular antioxidant systems, which include antioxidant vitamins, protein and non-protein thiols, and antioxidant enzymes. An acute bout of exercise at sufficient intensity has been shown to stimulate activities of antioxidant enzymes. This could be considered as a defensive mechanism of the cell under oxidative stress. However, we still have insufficient knowledge about the interaction between exercise and antioxidants, which are important in assessing the adequacy of protection against oxidative damage and about the necessity of dietary manipulation and/or supplementation. This review concerns effects of acute exercise on various oxidative stress parameters and antioxidant defense system.
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Introduction Restrictions on existing APOE mouse models have impacted research toward understanding the strongest genetic risk factor contributing to Alzheimer’s disease (AD) and dementia, APOE ε4 , by hindering observation of a key, common genotype in humans – APOE ε3/ε4 . Human studies are typically underpowered to address APOE ε4 allele risk as the APOE ε4/ε4 genotype is rare, which leaves human and mouse research unsupported to evaluate the APOE ε3/ε4 genotype on molecular and pathological risk for AD and dementia. Methods As a part of MODEL-AD, we created and validated new versions of humanized APOE ε3/ε3 and APOE ε4/ε4 mouse strains that, due to unrestricted breeding, allow for the evaluation of the APOE ε3/ε4 genotype. As biometric measures are often translatable between mouse and human, we profiled circulating lipid concentrations. We also performed transcriptional profiling of the cerebral cortex at 2 and 4 months (mos), comparing APOE ε3/ε4 and APOE ε4/ε4 to the reference APOE ε3/ε3 using linear modeling and WGCNA. Further, APOE mice were exercised and compared to litter-matched sedentary controls, to evaluate the interaction between APOE ε4 and exercise at a young age. Results Expression of human APOE isoforms were confirmed in APOE ε3/ε3 , APOE ε3/ε4 and APOE ε4/ε4 mouse brains. At two mos, cholesterol composition was influenced by sex, but not APOE genotype. Results show that the APOE ε3/ε4 and APOE ε4/ε4 genotype exert differential effects on cortical gene expression. APOE ε3/ε4 uniquely impacts ‘hormone regulation’ and ‘insulin signaling,’ terms absent in APOE ε4/ε4 data. At four mos, cholesterol and triglyceride levels were affected by sex and activity, with only triglyceride levels influenced by APOE genotype. Linear modeling revealed APOE ε3/ε4 , but not APOE ε4/ε4 , affected ‘extracellular matrix’ and ‘blood coagulation’ related terms. We confirmed these results using WGCNA, indicating robust, yet subtle, transcriptional patterns. While there was little evidence of APOE genotype by exercise interaction on the cortical transcriptome at this young age, running was predicted to affect myelination and gliogenesis, independent of APOE genotype with few APOE genotype-specific affects identified. Discussion APOE ε4 allele dosage-specific effects were observed in circulating lipid levels and cortical transcriptional profiles. Future studies are needed to establish how these data may contribute to therapeutic development in APOE ε3/ε4 and APOE ε4/ε4 dementia patients.
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