Physicochemical and metabolic basis for the differing neurotoxicity of the pyrrolizidine alkaloids,trichodesmine and monocrotaline

Monocrotaline and trichodesmine are structurally closely related pyrrolizidine alkaloids (PAs) exhibiting different extrahepatic toxicities, trichodesmine being neurotoxic (LD₅₀ 57 μmol/kg) and monocrotaline pneumotoxic (LD₅₀ 335 μmol/kg). We have compared certain physicochemical properties and metabolic activities of these two PAs in order to understand the quantitative and qualitative differences in toxicity. Both PAs were metabolized in the isolated, perfused rat liver to highly reactive pyrrolic dehydroalkaloids that appear to be responsible for the toxicity of PAs. More dehydrotrichodesmine (468 nmol/g liver) than dehydromonocrotaline (116 nmol/g liver) was released from liver into perfusate on perfusion for 1 hr with 0.5 mM of the parent PA. Dehydrotrichodesmine had a significantly longer aqueous half-life (5.4 sec) than that of dehydromonocrotaline (3.4 sec). In vivo, significantly higher levels of bound pyrroles were found in the brain 18 hr after injection of trichodesmine (25 mg/kg; i.p) than were seen following either an equal dose (25 mg/kg; i.p.) or an equitoxic dose (90 mg/kg; i.p.) of monocrotaline. Trichodesmine had a higher partition coefficient than monocrotaline for both chloroform and heptane, indicating its greater lipophilicity. The pK_a of trichodesmine (7.07) was only slightly higher than that of monocrotaline (pK_a 6.83), suggesting that a difference in degree of ionization was not a major factor affecting the relative ability of the dehydroalkaloids to cross the blood-brain barrier. We conclude that the greater lethality and neurotoxicity of trichodesmine compared to monocrotaline is due to two structural characteristics: (i) steric hindrance at position 14 of dehydrotrichodesmine results in greater resistance to hydrolysis, allowing more to be released from the liver and to be delivered to the brain; (ii) the larger isopropyl substituent at position 14 of dehydrotrichodesmine renders the molecule more lipophilic, leading to greater penetration of the brain. Special issue dedicated to Dr. Kinya Kuriyama     

钙调神经磷酸酶信号通路参与前列腺素F2α诱导的心肌肥厚

利用野百合碱（monocrotaline,MCT）诱导大鼠右心室肥厚模型和培养乳鼠心肌细胞,研究前列腺素F2α（prostaglandin F2α,PGF2α）在心肌肥厚中的作用及钙调神经磷酸酶（calcineurin,CaN）信号通路征其中的作用。在雄性Sprague-Dawley大鼠中,用MCT（60mg／kg）单次i．p．诱导右心室肥厚,同时用塞来旨布（20mg／kg）预防／治疗给药2周。用病理检测、电镜观察等方法观察心肌肥厚时组织病理改变;EIA试剂盒检测心肌组织PGF2α含量;RT-PCR检测心房钠尿肽（atrial natriuretic peptide,ANP）和CaNmRNA的表达;用蛋白免疫印迹法检测CaN及其下游因了NFAT3和GATA4蛋门质的表达。以心肌细胞直径、蛋白含量和ANP mRNA表达的变化为0．1μmol／L PGF2α诱导心肌细胞肥大的指标。以CaN mRNA表达作为该信号通路的主要指标,并观察CaN抑制剂环孢素A对PGF2α所致心肌细胞肥人和CaN mRNA表达的影响。结果显示：MCT注射2周（M2W组）,右心室肥厚指数（RVHI）、右心室／体重比及肺重／体重比分别增加了47％、53％和118％;注射后4周（M4W组）增加了64％、94％及156％。电镜观察发现右心室组织损伤。同时,右心室组织PGF2α含量在M2W和M4W组分别增加了44％和51％,与RVHI、ANP和CaN的mRNA表达,及CaN／NFAT3／GATA4的蛋白质表达均呈正相关。环氧酶抑制剂塞来昔布预防和治疗给药均明显改善MCT诱导的组织病理学改变。在高体细胞培养中,PGF2α（0．1μmol／L）明显使心肌细胞增大,蛋白质含量增加,ANP和CaN mRNA表达增强：同时,CaN抑制剂环孢素A明显抑制PGF2α诱导的心肌细胞肥大和CaN mRNA表达。上述结果提示：心肌组织局部PGF2α参与了MCT诱导的心肌肥厚过程,CaN信号通路是其细胞内信号转导通路之一。     

Astragaloside IV attenuates inflammatory response mediated by NLRP-3/calpain-1 is involved in the development of pulmonary hypertension

Inflammation eventually leads to pulmonary arterial hypertension (PAH). Astragaloside IV(AS-IV) has a protective effect on pulmonary hypertension, but the specific protective mechanism has been unclear until now. Therefore, in this study, our aim was to investigate the mechanisms underlying the effects of AS-IV on PAH. In vivo, male Sprague-Dawley (SD) rats were injected intraperitoneally with monocrotaline (MCT, 60 mg/kg) and treated with AS-IV (40 mg/kg, 80 mg/kg), MCC950 and MDL-28170. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with monocrotaline pyrrole (MCTP, 60 μg/mL). The protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1β and calpain-1 were measured in vivo and/or in vitro. The results showed that AS-IV decreased the protein expression levels of NLRP-3, caspase-1, ASC, IL-18, IL-1β and calpain-1 in vivo and/or vitro. In conclusion, in this study the results suggested that AS-IV could inhibit monocrotaline-induced pulmonary arterial hypertension via the NLRP-3/calpain-1 pathway.     

Melatonin attenuates monocrotaline-induced hepatic sinusoidal obstruction syndrome in rats via activation of Sirtuin-3

Melatonin possesses potent hepatoprotective properties, but it remains to be elucidated whether melatonin has a therapeutic effect on monocrotaline (MCT)-induced hepatic sinusoidal obstruction syndrome (HSOS). In this study, male Sprague Dawley rats were intraperitoneally injected with melatonin or the same volume of vehicle at 0 and 24 h after MCT intragastric administration. Next, hematoxylin–eosin staining and electron microscopy were performed to evaluate the hepatic sinusoidal injury of rats. Endothelial cell marker RECA-1 was observed by immunohistochemistry. Hepatic oxidative stress was analyzed by detecting malondialdehyde, glutathione S-transferase, and reactive oxygen species. Assessment of liver function was carried out by analysis of serum aspartate aminotransferase, alanine aminotransferase, total bilirubin, and albumin levels. Real-time polymerase chain reaction and Western blot analysis were used to identify liver Sirtuin-3 (SIRT3) and active matrix metallopeptidase 9 (MMP-9) expression. Besides, liver sinusoidal endothelial cells (LSECs) were used for the in vitro functional verification experiment. Specifically, liver histology of the melatonin-treated groups showed that the pathological damages caused by MCT were significantly attenuated, total HSOS scores were decreased, and the elevation of serum hyaluronic acid observed in the model group was also reduced. Moreover, melatonin treatment also improved the survival of rats after partial hepatectomy. Administration of melatonin ameliorated MCT-induced LSECs injury, hepatic oxidative stress, and hepatic dysfunction. Furthermore, melatonin treatment increased SIRT3 expression while attenuating MMP-9 activity in liver tissues. Cell experiment also demonstrated that SIRT3 might mediate the protective effect of melatonin on LSECs. Collectively, our study provided the potential rationale for the application of melatonin for the prevention of MCT-induced HSOS.     

POLYAMINE REGULATORY PROCESSES AND OXIDATIVE STRESS IN MONOCROTALINE-TREATED PULMONARY ARTERY ENDOTHELIAL CELLS

Alterations in polyamine metabolism may be a critical mechanism of monocrotaline (MCT)-induced structural remodeling of the pulmonary vasculature. In the present study, the hypothesis that MCT, through the induction of oxidative stress, modulates cellular polyamine regulatory mechanisms which in turn might be involved in the upregulation of fibronectin production in pulmonary artery endothelial cells (PAEC) was examined. A 24-h treatment with MCT significantly increased PAEC polyamine concentrations as compared to vehicle-treated cells. In addition, exposure to MCT caused an increase in abundance of ornithine decarboxylase (ODC) mRNA, upregulation of ODC activity and enhancement of spermidine import into PAEC. Inhibition ofde novopolyamine synthesis further increased spermidine uptake in MCT-treated cells. The depletion of cellular polyamine contents through the blockade of bothde novopolyamine biosynthesis and polyamine transport prevented MCT-induced increases in the medium level of fibronectin. In addition, PAEC treatment with MCT stimulated cellular oxidative stress as determined by increased levels of thiobarbituric acid reactive substances, enhanced dichlorofluorescein fluorescence and activation of NF-KB. A co-treatment with dimethylthiourea, an oxygen radical scavenger, prevented MCT-induced increases in cellular oxidation and attenuated disturbances in polyamine metabolism. These data suggest that MCT can stimulate polyamine regulatory processes in PAEC possibly through an increase in cellular oxidative stress. The present study may have significant implication in understanding mechanisms of MCT-induced pulmonary hypertension and remodeling of pulmonary vasculature.     

The involvement of Nrf2 antioxidant signalling pathway in the protection of monocrotaline-induced hepatic sinusoidal obstruction syndrome in rats by (+)-catechin hydrate

Hepatic sinusoidal obstruction syndrome (HSOS) is a rare and life-threatening liver disease. (+)-Catechin is a natural dietary flavonol with high antioxidant capacity. This study aims to investigate the involvement of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signalling pathway in the protection of (+)-catechin hydrate (CAT) against monocrotaline (MCT)-induced HSOS. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, total bilirubin (TBil) and bile acids (TBA) amounts, liver histological observation, scanning electron microscope evaluation, and hepatic metalloproteinase-9 (MMP-9) expression all demonstrated the protection of CAT against MCT-induced HSOS in rats. CAT attenuated MCT-induced liver oxidative injury in rats and the formation of cellular reactive oxygen species (ROS) in human hepatic sinusoidal endothelial cells (HHSECs). CAT-enhanced Nrf2 nuclear translocation in livers from MCT-treated rats and in HHSECs treated with MCT, and further increased the expression of Nrf2-dependent genes including catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLM), haem oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Moreover, GCL inhibitor _L-buthionine-(S, R)-sulfoximine (BSO), NQO1 inhibitor diminutol (Dim), and HO-1 inhibitor zinc protoporphyrin (ZnPP) all abrogated CAT-provided the protection against MCT-induced cytotoxicity in HHSECs. The results of molecular docking analysis indicated the potential interaction of CAT with the Nrf2-binding site in kelch-like ECH-associated protein-1 (Keap1) protein. In summary, this study demonstrated the critical involvement of Nrf2 antioxidant signalling pathway in CAT-provided the protection against MCT-induced HSOS.     

野百合碱诱导大鼠肺动脉高压及肺源性心脏病的病理生理特征

目的:探讨野百合碱诱发肺动脉高压及肺源性心脏病模型的建立机制。方法:雄性Wistar大鼠20只,随机分为两组(n=10):正常组,模型组。模型组大鼠腹腔一次性注射野百合碱50 mg/kg,对照组注射同剂量的溶媒,28 d后测定大鼠血流动力学参数,硝酸盐还原酶法测定血清和肺组织中一氧化氮的含量;放射免疫法测定血浆中内皮素、脑钠素和肺组织中肿瘤坏死因子、内皮素的含量。结果:与对照组比较,右心室压力上升、心率和平均动脉压下降,血液和肺组织中肿瘤坏死因子、一氧化氮、内皮素-1、脑钠素含量上升,具有统计学意义。结论:野百合碱通过诱发肺血管和组织炎性损伤,升高体内肿瘤坏死因子、一氧化氮、内皮素-1的含量,建立肺动脉高压及肺源性心脏病模型。     

Compensatory roles of Protein Related to DAN and Cerberus (PRDC) decrease in pulmonary arterial hypertension

Bone morphogenetic protein (BMP) signaling is commonly suppressed in patients with pulmonary arterial hypertension (PAH), but the compensatory mechanism of BMP signaling suppression is incompletely elucidated. This study aimed to investigate the role of PRDC, an antagonist of BMPs, in PAH and the underlying mechanism. Human lungs were collected and rat PAH was induced (monocrotaline, 60 mg/kg). BMP cascade and PRDC were detected in lungs and distal pulmonary artery smooth muscle cells (dPASMCs). In vitro cell experiments and in vivo supplementation of PRDC in hypertensive rats were subsequently performed. PRDC and BMP cascade all decreased in human and rat hypertensive lungs. Cell experiments confirmed that BMP2/4 inhibited dPASMCs proliferation by increasing cell cycle inhibitors (p21, p27), prevented dPASMCs migration by down-regulating MMP2/9 and up-regulating TIMP1/2 expression, and promoted dPASMCs apoptosis by up-regulating Bax, caspase3/9 and down-regulating Bcl-2 expression, as well as enhancing caspase3/7 activity, while, PRDC reversed the effects of BMP2/4 on dPASMCs proliferation, migration and apoptosis. In vivo trial found that PRDC supplementation deteriorated rat PAH in terms of pulmonary hemodynamics, vasculopathies and right ventricle hypertrophy. Taken together, compensatory decrease of PRDC in hypertensive lungs theoretically slow down the natural course of PAH, suggesting its therapeutic potential in PAH.     

银杏叶提取物对野百合碱诱导的大鼠右心室心肌肥厚的干预作用及其机制*

目的: 探讨银杏叶提取物对大鼠右心室心肌肥厚的干预作用及其机制。方法: 72 只SD 大鼠随机分为3 组,每组24只: 对照组(CON组)、野百合碱诱导右心室心肌肥厚组(MCT组)、银杏叶提取物干预组(EGB组)。MCT组与EGB组首日均以2%MCT按60 mg/kg 剂量腹腔注射,注射后第2日开始,MCT组每日予2 ml 0.9% NaCl灌胃,EGB组以60 mg/kg银杏叶提取物灌胃,对照组SD大鼠首日一次性腹腔注射2 ml 0.9%NaCl注射液。3周后检测各组大鼠心脏血流动力学变化、计算心脏重量指数、HE染色观察心肌病理形态学变化、RT-PCR法检测TRPC6 mRNA表达和Western blot法检测蛋白的表达水平。结果: 与对照组比较,MCT组反映右心室肥厚程度的指标如RVSP、RV±dp/dt_max及RVMI显著增加(P＜0.01),而EGB早期干预组虽然与MCT组的各项指标有相同趋势的变化,但是EGB组各项指标变化的幅度均显著降低(P＜0.01),且EGB组的心肌肥厚指数均显著低于MCT组(P＜ 0.01);HE染色观察心肌形态学变化结果:MCT组呈典型心肌肥厚表现;EGB组右心室心肌细胞较MCT组有显著改善;MCT组及EGB组SD大鼠右心室TRPC6 mRNA及蛋白相对表达水平升高(P＜0.05),而EGB组较MCT组显著降低(P＜0.05)。结论: 银杏叶提取物可能通过降低TRPC6的表达阻碍心肌细胞中CaN／NFAT信号路径而发挥对心肌肥厚的早期保护作用。     

Role of the gap junctions in the contractile response to agonists in pulmonary artery from two rat models of pulmonary hypertension

Background

Pulmonary hypertension (PH) is characterized by arterial vascular remodelling and alteration in vascular reactivity. Since gap junctions are formed with proteins named connexins (Cx) and contribute to vasoreactivity, we investigated both expression and role of Cx in the pulmonary arterial vasoreactivity in two rat models of PH.

Methods

Intrapulmonary arteries (IPA) were isolated from normoxic rats (N), rats exposed to chronic hypoxia (CH) or treated with monocrotaline (MCT). RT-PCR, Western Blot and immunofluorescent labelling were used to study the Cx expression. The role of Cx in arterial reactivity was assessed by using isometric contraction and specific gap junction blockers. Contractile responses were induced by agonists already known to be involved in PH, namely serotonin, endothelin-1 and phenylephrine.

Results

Cx 37, 40 and 43 were expressed in all rat models and Cx43 was increased in CH rats. In IPA from N rats only, the contraction to serotonin was decreased after treatment with ^37-43Gap27, a specific Cx-mimetic peptide blocker of Cx 37 and 43. The contraction to endothelin-1 was unchanged after incubation with ⁴⁰Gap27 (a specific blocker of Cx 40) or ^37-43Gap27 in N, CH and MCT rats. In contrast, the contraction to phenylephrine was decreased by ⁴⁰Gap27 or ^37-43Gap27 in CH and MCT rats. Moreover, the contractile sensitivity to high potassium solutions was increased in CH rats and this hypersensitivity was reversed following ^37-43Gap27 incubation.

Conclusion

Altogether, Cx 37, 40 and 43 are differently expressed and involved in the vasoreactivity to various stimuli in IPA from different rat models. These data may help to understand alterations of pulmonary arterial reactivity observed in PH and to improve the development of innovative therapies according to PH aetiology.     

Deficiency of cold-inducible RNA-binding protein exacerbated monocrotaline-induced pulmonary artery hypertension through Caveolin1 and CAVIN1

Cold-inducible RNA-binding protein (CIRP) was a crucial regulator in multiple diseases. However, its role in pulmonary artery hypertension (PAH) is still unknown. Here, we first established monocrotaline (MCT)-induced rat PAH model and discovered that CIRP was down-regulated predominantly in the endothelium of pulmonary artery after MCT injection. We then generated Cirp-knockout (Cirp-KO) rats, which manifested severer PAH with exacerbated endothelium damage in response to MCT. Subsequently, Caveolin1 (Cav1) and Cavin1 were identified as downstream targets of CIRP in MCT-induced PAH, and the decreased expression of these two genes aggravated the injury and apoptosis of pulmonary artery endothelium. Moreover, CIRP deficiency intensified monocrotaline pyrrole (MCTP)-induced rat pulmonary artery endothelial cells (rPAECs) injuries both in vivo and in vitro, which was counteracted by Cav1 or Cavin1 overexpression. In addition, CIRP regulated the proliferative effect of conditioned media from MCTP-treated rPAECs on rat pulmonary artery smooth muscle cells, which partially explained the exceedingly thickened pulmonary artery intimal media in Cirp-KO rats after MCT treatment. These results demonstrated that CIRP acts as a critical protective factor in MCT-induced rat PAH by directly regulating CAV1 and CAVIN1 expression, which may facilitate the development of new therapeutic targets for the intervention of PAH.     

TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

Disease severity–related alterations of cardiac microRNAs in experimental pulmonary hypertension

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart‐relevant microRNAs, that is myomiRs (miR‐1, miR‐133a, miR‐208, miR‐499) and miR‐214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT‐qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR‐1/miR‐133a/miR‐208a/miR‐499) were decreased and the expression of miR‐214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR‐214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.     

Evidence for a pro-calcitonin

The biosynthesis of calcitonin was studied using radioimmunochemical methods and suspensions of calcitonin-producing cells derived from trout ultimobranchial glands. [¹⁴C]leucine was incorporated into cell proteins in a linear fashion for up to 36 hrs. Acid-extracted cellular radioactivity could be precipitated by trichloroacetic acid and calcitonin antiserum. Chromatography of the cell extracts revealed two distinct peaks of radio-immunoassayable and immunoprecipitable calcitonin activity. One peak coeluted with radioiodinated calcitonin, the other as a higher molecular weight species. The relative incorporation of [¹⁴C]leucine into the higher and lower molecular weight peaks during “pulse-chase” experiments was consistent with a precursor-product relationship between them.     

A mechanism for setting a single rhythm for a multipacemaker sinoatrial node

Interaction of the system of n pacemaker cells modelling the work of the heart sinoatrial node was studied. Suggesting the interaction additivity an expression was obtained for the system single rhythm. Dependence of the system single rhythm and propagation velocity of excitation delay on the number of pacemakers of the leading centre and connection force between the pacemakers was investigated. The results obtained qualitatively, agree with the experimental evidence available.