DL-3-n-butylphthalide alleviates motor disturbance by suppressing ferroptosis in a rat model of Parkinson’s disease

DL-3-n-butylphthalide(NBP)-a compound isolated from Apium graveolens seeds-is protective against brain ischemia via various mechanisms in humans and has been approved for treatment of acute ischemic stroke.NBP has shown recent potential as a treatment for Parkinson’s disease.However,the underlying mechanism of action of NBP remains poorly understood.In this study,we established a rat model of Parkinson’s disease by intraperitoneal injection of rotenone for 28 successive days,followed by intragastric injection of NBP for 14-28 days.We found that NBP greatly alleviated rotenone-induced motor disturbance in the rat model of Parkinson’s disease,inhibited loss of dopaminergic neurons and aggregation ofα-synuclein,and reduced iron deposition in the substantia nigra and iron content in serum.These changes were achieved by alterations in the expression of the iron metabolism-related proteins transferrin receptor,ferritin light chain,and transferrin 1.NBP also inhibited oxidative stress in the substantia nigra and protected mitochondria in the rat model of Parkinson’s disease.Our findings suggest that NBP alleviates motor disturbance by inhibition of iron deposition,oxidative stress,and ferroptosis in the substantia nigra.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

DL-3-n-butylphthalide improved physical and learning and memory performance of rodents exposed to acute and chronic hypobaric hypoxia

Background:Studies have revealed the protective effect of DL-3-n-butylphthalide(NBP)against diseases associated with ischemic hypoxia.However,the role of NBP in animals with hypobaric hypoxia has not been elucidated.This study investigated the effects of NBP on rodents with acute and chronic hypobaric hypoxia.Methods:Sprague-Dwaley rats and Kunming mice administered with NBP(0,60,120,and 240 mg/kg for rats and 0,90,180,and 360 mg/kg for mice)were placed in a hypobaric hypoxia chamber at 10,000 m and the survival percentages at 30 min were determined.Then,the time and distance to exhaustion of drug-treated rodents were evaluated during treadmill running and motor-driven wheel-track treadmill experiments,conducted at 5800 m for 3 days or 20 days,to evaluate changes in physical functions.The frequency of active escapes and duration of active escapes were also determined for rats in a shuttle-box experiment,conducted at 5800 m for 6 days or 27 days,to evaluate changes in learning and memory function.ATP levels were measured in the gastrocnemius muscle and malonaldehyde(MDA),superoxide dismutase(SOD),hydrogen peroxide(H_(2)O_(2)),glutathione peroxidase(GSH-Px),and lactate were detected in sera of rats,and routine blood tests were also performed.Results:Survival analysis at 10,000 m indicated NBP could improve hypoxia tolerance ability.The time and distance to exhaustion for mice(NBP,90 mg/kg)and time to exhaustion for rats(NBP,120 and 240 mg/kg)significantly increased under conditions of acute hypoxia compared with control group.NBP treatment also significantly increased the time to exhaustion for rats when exposed to chronic hypoxia.Moreover,240 mg/kg NBP significantly increased the frequency of active escapes under conditions of acute hypoxia.Furthermore,the levels of MDA and H_(2)O_(2) decreased but those of SOD and GSH-Px in the sera of rats increased under conditions of acute and chronic hypoxia.Additionally,ATP levels in the gastrocnemius muscle significantly increased,while lactate levels in sera significantly decreased.Conclusion:NBP improved physical and learning and memory functions in rodents exposed to acute or chronic hypobaric hypoxia by increasing their anti-oxidative capacity and energy supply.

EFFECT OF DL-3-N-BUTYLPHTHALIDE ON BRAIN EDEMA IN RATS SUBJECTED TO FOCAL CEREBRAL ISCHEMIA

The present study evaluated the effect of dl-3-n-butylphthalide(NBP) ,a novel brain protective agent, on brain edema in rats following focal ischemia. Edema was induced by occluding the right middle cerebral artery (MCAO).producing permanent focal ischemia in the right cerebral hemisphere,which developed ip-silateral brain edema reproducibly. Edema was assessed 24 h after MCA occlusion by determining the brain water content from wet and dry weight measurements,and the sodium,potassium concentrations with ion-selective electrodes. In this model,NBP at the dose of 80,160 and 240 mg/kg po 15 min after MCAO prevented from brain edema in a dose-dependent manner. A significant reduction of sodium content and an increase in potassium level were observed in all drug-treated groups. It showed that NBP strongly attenuated brain water entry,sodium accumulation and potassium loss. Nimodipine treatment(5mg/kg sc) also reduced brain edema (P<0. 05). The results suggest that a strong anti-edema activity of NBP may play an important role to contribute to the treatment of ischemic damage.

DI-3-n-butylphthalide exerts neuroprotective effects by modulating hypoxia-inducible factor 1-alpha ubiquitination to attenuate oxidative stress-induced apoptosis

DI-3-n-butylphthalide is used to treat mild and moderate acute ischemic stroke.However,the precise underlying mechanism requires further investigation.In this study,we investigated the molecular mechanism of DI-3-n-butylphthalide action by various means.We used hydrogen peroxide to induce injury to PC12cells and RAW264.7 cells to mimic neuronal oxidative stress injury in stroke in vitro and examined the effects of DI-3-n-butylphthalide.We found that DI-3-nbutylphthalide pretreatment markedly inhibited the reduction in viability and reactive oxygen species production in PC12 cells caused by hydrogen peroxide and inhibited cell apoptosis.Furthermore,DI-3-n-butylphthalide pretreatment inhibited the expression of the pro-apoptotic genes Bax and Bnip3.DI-3-nbutylphthalide also promoted ubiquitination and degradation of hypoxia inducible factor 1α,the key transcription factor that regulates Bax and Bnip3 genes.These findings suggest that DI-3-n-butylphthalide exhibits a neuroprotective effect on stroke by promoting hypoxia inducible factor-1α ubiquitination and degradation and inhibiting cell apoptosis.

Ninety-day administration of dl-3-n-butylphthalide for acute ischemic stroke： a randomized, double-blind trial

Background DI-3-n-butylphthalide （NBP）, first isolated from the seeds of celery, showed efficacy in animal models of stroke. This study was a clinical trial to assess the efficacy and safety of NBP with a continuous dose regimen among patients with acute ischemic stroke. Methods A randomized, double-blind, double-dummy trial enrolled 573 patients within 48 hours of onset of ischemic stroke in China. Patients were randomly assigned to receive a 14-day infusion of NBP followed by an NBP capsule, a 14- day infusion of NBP followed by aspirin, or a 14-day infusion of ozagrel followed by aspirin. The efficacy measures were Barthel index score and the modified Rankin scale （mRS） at day 90. Differences among the three groups on mRS were compared using X2 test of proportions （with two-sided e=0.05） and Logistic regression analysis was conducted to take the baseline National Institutes of Health Stroke Scale （NIHSS） score into consideration. Results Among the 535 subjects included in the efficacy analysis, 90-day treatment with NBP was associated with a significantly favorable outcome than 14-day treatment with ozagrel as measured by mRS （P 〈0.001）. No significant difference was found among the three groups on Barthel index at day 90. The rate of adverse events was similar among the three groups. Conclusions The 90-day treatment with NBP could improve outcomes at the third month after stroke. The NBP treatment （both intravenous and oral） is safe （ChiCTR-TRC-09000483）.

DL-3-n-butylphthalide protects the blood-brain barrier against ischemia/hypoxia injury via upregulation of tight junction proteins

Background:The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia. Methods: A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1%〇2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3p (GSK-3(3)/GSK-3p, and (3-catenin/p-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy. Results: In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 (μg/g vs. 12.3 ± 1.9 (μg/g, P = 0.005;6.7 ± 0.6 μg/g vs. 12.3 ± 1.9μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 mvs. 0.41 ± 0.06, P < 0.01;0.07 ± 0 .0 7 vs. 0.41 ± 0 .0 6 , P < 0 .61 ), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with the corresponding values in the hypoxia group. NBP treatment also elevated the relative expression levels of p-Akt/Akt, p-GSK-3p/GSK-3β, and β-catenin/β-actin in comparison with the corresponding values in the hypoxia group (all P < 0 .0 5 ). Conclusion: NBP improves the barrier function of BBB against ischemic injury by upregulating the expression of TJ proteins, possibly by reducing oxidative stress and activating the Akt/GSK-3β/β-catenin signaling pathway.

Protective effects of dl-3-n-butylphthalide on changes of regional cerebral blood flow and blood-brain barrier damage following experimental subarachnoid hemorrhage

ｄｌ３ｎｂｕｔｙｌｐｈｔｈａｌｉｄｅ（ｄｌＮＢＰ）ｉｓａｎｅｗｃａｎｄｉｄａｔｅｆｏｒｔｒｅａｔｍｅｎｔｏｆｃｅｒｅｂｒａｌｉｓｃｈｅｍｉａ．ＯｕｒｐｒｅｖｉｏｕｓｓｔｕｄｉｅｓｓｈｏｗｅｄｔｈａｔｄｌＮＢＰｉｓａｂｌｅｔｏｒｅｄｕｃｅｔｈ...

Therapeutic effects of dl-3-n-butylphthalide in a transgenic mouse model of amyotrophic lateral sclerosis

Background Amyotrophic lateral sclerosis （ALS） is a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Transgenic mice over-expressing a mutant form of the human SOD1 gene develop an ALS-like phenotype. Currently, there is no effective treatment or drug for the fatal disease. Previous studies reported potent efficacy of dl-3-n-butylphthalide （DL-NBP） for several neurodegenerative disorders and cerebral ischemia. SOD1-G93A mice are a mouse model of ALS. In this study, we investigated the efficacy of DL-NBP on this ALS mouse model. Methods Sixty SOD1-G93A female mice were divided into four groups. The vehicle control group received 0 mg.kg-1.d-~ DL-NBP. The experimental groups received DL-NBP with doses of 30, 60 or 120 mg.kgl.d1, respectively. For measurement of motor activity, the hanging wire test and rotarod test were performed. Survival statistics were analyzed by Kaplan-Meier survival curves. The body weight of each mouse was recorded twice per week. The statistical motor unit number estimation （MUNE） technique was used to estimate the number of functioning motor units in gastrocnemius muscle. Muscle morphology was evaluated by hematoxylin and eosin staining. Motor neuron quantJtation was performed by Nissl staining and microglia activation was observed by immunohistochemistry. Results Oral administration of 60 mg.kg-l-d-1 DL-NBP significantly prolonged survival （（164.78±16.67） days） of SOD1-G93A mice compared with vehicle control （（140.00＋16.89） days）. Treating mice with DL-NBP （60 mg.kg-1.d-1） significantly decreased the progression rate of motor deficits and suppressed body weight reduction. Furthermore, we found that treating SOD1-G93A mice with DL-NBP （60 mg.kgl.d1） slowed the rate of MUNE reduction （P 〈0.01）. Motor neurons were remarkably preserved in the anterior horns in mice treated with DL-NBP （60 mg.kg-1d-1） at the stage of 19 weeks （P 〈0.01）. Treating mice with DL-NBP （60 mg.kg1.d1） significantly reduced CD11b immunoreactivity compared with vehicle control mice （P 〈0.05）. No significant effect was observed in mice treated with DL-NBP of 30 or 120 mg.kg-1.d-1. Conclusions The post-disease-onset administration of DL-NBP significantly prolonged survival and improved motor performance in SOD1-G93A mice. DL-NBP mav be a Dotential theraDeutic aaent for ALS.

3-N-Butylphthalide mitigates high glucose-induced injury to Schwann cells:association with nitrosation and apoptosis

A high glucose state readily causes peripheral axon atrophy, demyelination, loss of nerve fiber function, and delayed regeneration. However, few studies have examined whether nitration is also critical for diabetic peripheral neuropathy. Therefore, this study investigated the effects of high glucose on proliferation, apoptosis, and 3-nitrotyrosine levels of Schwann cells treated with butylphthalide. In addition, we explored potential protective mechanisms of butylphthalide on peripheral nerves. Schwann cells were cultured in vitro with high glucose then stimulated with the peroxynitrite anion inhibitors uric acid and 3-n-butylphthalide for 48 hours. Cell Counting Kit-8 and flow cytometry were used to investigate the effects of uric acid and 3-n-butylphthalide on proliferation and apoptosis of Schwann cells exposed to a high glucose environment. Effects of uric acid and 3-n-butylphthalide on levels of 3-nitrotyrosine in Schwann cells were detected by enzyme-linked immunosorbent assay. The results indicated that Schwann cells cultured in high glucose showed decreased proliferation, but increased apoptosis and intracellular 3-nitrotyrosine levels. However, intervention with uric acid or 3-n-butylphthalide could increase proliferation of Schwann cells cultured in high glucose, and inhibited apoptosis and intracellular 3-nitrotyrosine levels. According to our data, 3-n-butylphthalide may inhibit cell nitrification and apoptosis, and promote cell proliferation, thereby reducing damage to Schwann cells caused by high glucose.

L-3-n-butylphthalide protects against vascular dementia via activation of the Akt kinase pathway

As a neuroprotective drug for the treatment of ischemic stroke, 3-n-butylphthalide, a celery seed ex- tract, has been approved by the State Food and Drug Administration of China as a clinical therapeutic drug for ischemic stroke patients. L-3-n-butylphthalide possesses significant efficacy in the treatment of acute ischemic stroke. The activated Akt kinase pathway can prevent the death of nerve cells and exhibit neuroprotective effects in the brain after stroke. This study provides the hypothesis that I-3-n- butylphthalide has a certain therapeutic effect on vascular dementia, and its mechanism depends on the activation of the Akt kinase pathway. A vascular dementia mouse model was established by cere- bral repetitive ischemia/reperfusion, and intragastrically administered I-3-n-butylphthalide daily for 28 consecutive days after ischemia/repedusion, or 7 consecutive days before ischemia/reperfusion. The Morris water maze test showed significant impairment of spatial learning and memory at 4 weeks after operation, but intragastric administration of I-3-n-butylphthalide, especially pretreatment with I-3-n- butylphthalide, significantly reversed these changes. Thionine staining and western blot analylsis showed that preventive and therapeutic application of I-3-n-butylphthalide can reduce loss of pyrami- dal neurons in the hippocampal CA1 region and alleviate nerve damage in mice with vascular demen- tia. In addition, phosphorylated Akt expression in hippocampal tissue increased significantly after I-3-n- butylphthalide treatment. Experimental findings demonstrate that I-3-n-butylphthalide has preventive and therapeutic effects on vascular dementia, and its mechanism may be mediated by upregulation of phosphorylated Akt in the hippocampus.

Effects of L-3-n-butylphthalide on caspase-3 and nuclear factor kappa-B expression in primary basal forebrain and hippocampal cultures after beta-amyloid peptide 1-42 treatment

BACKGROUND： L-3-n-butylphthalide （L-NBP） can inhibit phosphorylation of tau protein and reduce the neurotoxicity of beta-amyloid peptide 1-42 （Aβ1-42）. OBJECTIVE： To observe the neuroprotective effects of L-NBP on caspase-3 and nuclear factor kappa-B （NF- K B） expression in a rat model of Alzheimer＇s disease. DESIGN, TIME AND SETTING： A cell experiment was performed at the Central Laboratory of Provincial Hospital affiliated to Shandong University between January 2008 and August 2008. MATERIALS： L-NBP （purity 〉 98%） was provided by Shijiazhuang Pharma Group NBP Pharmaceutical Company Limited. Aβ1-42, 3-[4,5-dimethylthiazolo-2]-2,5 iphenyltetrazolium bromide （MTT）, and rabbit anti-Caspase-3 polyclonal antibody were provided by Cell Signaling, USA; goat anti-choactase and rabbit anti-NF- kB antibodies were provided by Santa Cruz, USA. METHODS： Primary cultures were generated from rat basal forebrain and hippocampal neurons at 17 or 19 days of gestation. The cells were assigned into five groups： the control group, the Aβ1-42 group （2 μmol/L）, the Aβ1-42 ＋ 0.1 μmol/L L-NBP group, the Aβ1-42 ＋ 1 μ mol/L L-NBP group, and the Aβ1-42 ＋ 10μmol/L L-NBP group. The neurons were treated with Aβ1-42 （2 μmol/L） alone or in combination with L-NBP （0.1, 1, 10 μmol/L） for 48 hours. Cells in the control group were incubated in PBS. MAIN OUTCOME MEASURES： Morphologic changes were evaluated using inverted microscopy, viability using the M-I-I- method, and the changes in caspase-3 and NF- k B expression using Western blot. RESULTS： Induction with Aβ1-42 for 48 hours caused cell death and soma atrophy, and increased caspase-3 and NF- K B expression （P 〈 0.05）. L-NBP blocked these changes in cell morphology, decreased caspase-3 and NF- k B expression （P 〈 0.05）, and improved cell viability, especially at the high dose （P 〈 0.05）. CONCLUSION： AI3^-42 is toxic to basal forebrain and hippocampal primary neurons; L-NBP protects against this toxicity and inhibits the induction of caspase-3 and NF- K B expression.

Study on the Mechanism of the Rearrangement Reaction of 3-n-Butylphthalide by Deuterium-Labelling

By the Grignard reaction of 1,1-di-deutero-1-bromobutane with phthalaldehydic acid 1',1'-di-deutero-3-n-butyl phthalide was obtained, which underwent a rearrangement reaction using AlCl3 as catalyst in CS2 to give 1-methyl-5-carboxy-3,4-di-deutero-tetrahydronaphthalene. The mechanism was proposed to be a series of consecutive 1,2- hydride transfers rather than a direct 1,4-hydride transfer.

依达拉奉联合Dl-3-正丁基苯酞软胶囊治疗进展型脑梗死40例临床观察

将同期收治的120例进展型脑梗死患者随机分为A、B、C组各40例,三组均予低分子右旋糖酐、胞二磷胆碱等常规治疗,在此基础上A组予依达拉奉静滴、Dl-3-正丁基苯酞软胶囊口服;B组静滴依达拉奉,方法剂量同上;C组予复方丹参静滴,均14 d为一疗程。观察三组疗效及症状评分。结果A、B、C组显效率均显著高于C组(P<0.05);治疗后症状评分均显著低于治疗前及C组,A组低于B组。认为依达拉奉联合Dl-3-正丁基苯酞软胶囊对进展型脑梗死有协同治疗效果。

HCHO-(DL-苹果酸)-BrO_3^--Mn^(2+)H_2SO_4体系化学振荡反应动力学研究

研究了HCHO参与下的(DL 苹果酸) BrO-3 Mn2+ H2SO4化学振荡反应体系的非线性动力学行为,考察了该体系中各反应物的初始浓度范围及主要影响因素.结果表明,在5.0×10-5～1.0×10-2mol·L-1范围内,HCHO对振荡反应的诱导期和周期有较大影响,且HCHO浓度的对数lnc(HCHO)与诱导期倒数的对数ln(1/tin)及周期倒数的对数ln(1/tp)均存在线性关系.诱导期和周期的表观活化参数分别为70.87,55.71kJ·mol-1.另外还对HCHO参与下的可能振荡反应机理进行了探讨.

手性酒石酸酯对不对称环氧化反应动力学拆分DL-1-十四烯-3-醇的催化选择性

利用Sharpless不对称环氧化反应动力学拆分二级烯丙醇DL 1 十四烯 3 醇 ,研究了不同手性酒石酸酯催化剂 [酒石酸乙酯 (DET)、酒石酸异丙酯 (DIPT)、酒石酸环己酯 (DCHT)以及酒石酸环十二酯 (DCDT) ]对该反应的催化选择性 ,拆分所得的烯丙醇的光学纯度有一定差异 ,从 90 %到 99%ee .

dl-3-正丁基苯酞软胶囊治疗急性进展性脑梗死疗效观察

目的观察dl-3-正丁基苯酞软胶囊治疗急性进展性脑梗死的疗效。方法将60例脑梗死患者随机分为治疗组和对照组,各30例。对照组给予血栓通加入生理盐水静脉输注,每日1次。治疗组在此基础上给予丁苯酞软胶囊口服,疗程为2周。评价两组患者在治疗前及治疗后第7天、第14天的神经功能缺损程度。结果治疗组治疗第7天和第14天的临床神经功能缺损程度评分较治疗前均显著降低(P<0.05),且与对照组治疗后比较差异有统计学意义(P<0.05)。结论 dl-3-正丁基苯酞软胶囊对急性进展性脑梗死的治疗具有显著效果。

多巴丝肼联用dl-3-正丁基苯酞对脑卒中患者患侧上肢功能的影响

目的观察多巴丝肼联用dl-3-正丁基苯酞对脑卒中患者患侧上肢功能恢复的影响。方法 90例脑卒中偏瘫患者采用随机数字表法分为康复组、治疗组及对照组,每组30例。康复组和治疗组患者均给予运动疗法治疗,其中治疗组患者加用口服多巴丝肼、dl-3-正丁基苯酞治疗。分别于治疗前、治疗8周时采用运动功能评分(FMA)、运动功能评估量表(MAS)对患者上肢功能进行评定。结果治疗前患者上肢FMA、MAS评分差异无显著性;治疗8周后,康复组和治疗组患者上肢FMA及MAS评分均较治疗前改善,治疗组较康复组改善,且均优于对照组。结论多巴丝肼联用dl-3-正丁基苯酞胶囊可进一步改善患者上肢运动功能。