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.

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.

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.

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

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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.