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Objective To elucidate the mechanism of dl-3-N-butylphthalide (NBP) on renal ischemia-reperfusion injury (IRI) in rat models. Methods Forty SD rats were randomly divided into the sham operation group (Sham group), model group (IRI group), NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), low-dose NBP group (NBP-L group) and high-dose NBP group (NBP-H group), with 8 rats in each group. Serum creatinine (Scr), serum cystatin C(Cys-C), blood urea nitrogen (BUN) and serum interleukin (IL)-1β and IL-18 levels were detected in all groups. Pathological injury of renal tissues in each group was observed by Hematoxylin-eosin (HE) staining. The expression levels of inflammatory factors and nuclear factor (NF)-κB signaling pathway and cell pyroptosis-related proteins in renal tissues were measured by Western blot and immunohistochemical staining. Results Compared with the Sham group, renal tissue injury was more severe, and the levels of Scr, Cys-C, BUN and serum IL-1β and IL-18 were all up-regulated in the IRI group. Western blot showed that the relative expression levels of NOD-like receptor protein (NLRP3), Gasdermin D(GSDMD), cysteinyl aspartate specific proteinase (Caspase)-1, IL-18, IL-1β, NF-κB p65 and p-NF-κB p65 proteins were all up-regulated, and immunohistochemical staining revealed that the expression levels of NF-κB p65 and p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were all up-regulated in the IRI group. Compared with the IRI group, renal tissue injury was alleviated, and the levels of Scr, Cys-C, BUN and serum IL-18 and IL-1β were down-regulated in the PDTC, NBP-L and NBP-H groups. Western blot showed that the expression levels of NLRP3, GSDMD, Caspase-1, IL-1β, IL-18, NF-κB p65 and p-NF-κB p65 proteins were down-regulated, and immunohistochemical staining indicated that the expression levels of NF-κB p65, p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were down-regulated in the PDTC, NBP-L and NBP-H groups, respectively. Compared with the NBP-L group, renal tissue injury was mitigated, and the levels of Scr, Cys-C, BUN, serum IL-18 and IL-1β were all down-regulated in the NBP-H group. Western blot showed the expression levels of NLRP3, GSDMD, Caspase-1, IL-1β, IL-18, NF-κB p65 and p-NF-κB p65 proteins were down-regulated in the NBP-H group. Immunohistochemical staining indicated that the expression levels of NF-κB p65, p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were down-regulated in the NBP-H group. Conclusions NBP may down-regulate the activity of NF-κB/NLRP3 signaling pathway and reduce the expression levels of cell pyroptosis-related proteins and inflammatory factors after renal IRI, thereby suppressing cell pyroptosis and alleviating renal IRI.
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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 1.0 μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1% O2, 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 3β (GSK-3β)/GSK-3β, and β-catenin/β-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 ± 1.9 μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 vs. 0.41 ± 0.06, P < 0.01; 0.97 ± 0.07 vs. 0.41 ± 0.06, P < 0.01), 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-3β/GSK-3β, and β-catenin/β-actin in comparison with the corresponding values in the hypoxia group (all P < 0.05).@*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.
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6-Bromo-3-n-butylphthalide was obtained by nitration, reduction and diazotization from carboxybenzaldehyde. Twenty hybrids from substituted styrene and 6-bromo-3-n-butylphthalide were synthesized and the structure was confirmed by 1H NMR, 13C NMR and ESI-MS. All compounds were evaluated for neuroprotective activity against OGD/R-induced neurotoxicity in rat cortical neurons by MTT assay. The mechanism of neuroprotection was investigated by Western blot analyses. The results indicated that most of these compounds had a potent neuroprotective activity (All animal experiments were approved by the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine), especially 10h and 10i showed significant effects, which may play a neuroprotective role by activating the PI3K/Akt signaling pathway.
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Objective To investigate the effects of DL-3-n-Butylphthalide(NBP)on proliferation and apoptosis of 1-methyl-4-phenyl-pyridinium (MPP +)-induced SH-SY5Y cells, and mechanisms via mixed lineage kinase 3 (MLK3) signaling pathway. Methods The SH-SY5Y cells were divided into control group,MPP+group,NBP group and URMC-099 group,that cultured normally,with 1 mmol/L MPP+for 24 hours,with 10μmol/L NBP for 3 hours and then with MPP+for 24 hours,and with 200 nmol/L MLK3 inhibitor URMC-099 for 3 hours and then with MPP+for 24 hours,respectively.The morphology of SH-SY5Y cells was observed under inverted phase contrast mi-croscope and the survival rate was measured with 3-(4,5-Cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays.The apoptosis was quantified under flow cytometry with Annexin V/PI fluorescence staining,and the nuclear morphology was observed with Hoechst 33342 staining.The expression of phosphorylated protein of MLK3(p-MLK3),c-Jun N-terminal kinase(p-JNK),extra cellular regulated protein ki-nases(p-ERK1/2)were detected with Western blotting.Results Compared with the control group,the survival rate reduced and apoptosis in-creased in MPP+group(P<0.05),with the increase of p-MLK3 and p-JNK and decrease of p-ERK1/2 d(P<0.05).Compared with MPP+group,the survival rate increased and apoptosis reduced in both NBP and URMC-099 groups(P<0.05),with the decrease of p-MLK3 and p-JNK and increase of p-ERK1/2(P<0.05).Conclusion NBP can decrease the apoptosis and promote the proliferation of SH-SY5Y cells in-duced by MPP+,which may be associated with inhibiting MLK3 signaling pathway,and regulating the downstream p-JNK and p-ERK1/2.
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Objective To investigate the effect of Dl-3-n-butylphthalide on acute cerebral infarction.Methods In Department of Neurology in the Fifth Hospital of Wuhan from March 2013 to June 2014,100 cases of patients with first onset of acute cerebral infarction were recruited.The participants were divided into 2 groups (control group and treatment group) randomly,with 50 participants in each group.Besides general treatment,the patients of treatment group received intravenous injection of Dl-3-n-butylphthalide in acute phase and orally took soft capsule of Dl-3-n-butylphthalide in recovery phase.All the patients were followed up for 24 weeks.Neurological function and general cognition were assessed separately by national institute of health stroke scale (NIHSS),and mini mental state examination (MMSE) was applied to assess overall cognitive function.Results NHISS score was gradually decreased and MMSE score was increased in both groups.As compared with the control group,NIHSS score and MMSE score were changed significantly in the treatment group.From first onset to 24 weeks after treatment,NHISS score was decreased by 30％ in the control group and 44％ in the treatment group;MMSE score was increased by 17％ in the control group and 32％ in the treatment group.Conclusion Sequential therapy with Dl-3-n-butylphthalide improves neurological function and general cognition faster and more significant for patients with acute cerebral infarction.
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Objective:To probe the therapeutic effects of Butylphthalide Injection in the elderly patients with acute cerebral infarction (ACI) and its influence in cerebral hemodynamics and cerebral vascular reserve (CVR),and to clarify the pharmacological action mechanism of butylphthalide in treatment of ACI.Methods:A total of 100 cases of elderly patients with ACI were selected as the subjects and divided into observation group and control group according to the serial number on admission.Fifty cases were included in each group.The patients in control group were treated with the conventional treatment, while the patients in observation group were treated with Butylphthalide Injection on the basis of the conventional treatment.The National Institute of Health Stroke Scale (NIHSS) score, the brain hemodynamics indexes of the peak velocity (Vp), the mean velocity (Vm) and the differences of the velocity (DVp, DVm) as well as pulsatility index (PI), CVR of bilateral middle cerebral artery (MCA)of the patients in two groups were observed and compared.The therapeutic effects of the patients in two groups were evaluated and compared.Results:The NIHSS score of the patients in observation group after treatment was significantly lower than that in control group (t=15.420, P<0.05).The therapeutic effects and the clinical efficiency of the patients in observation group were significantly better than those in control group (U=2.225, χ2=5.005, P<0.05).The Vp and Vm of the patients in observation group after treatment were significantly higher than those in control group(t=10.819,t=7.259, P<0.05)and the DVp and DVm were significantly lower than those in control group (t=16.438,t=19.055, P<0.05).The CVR of the patients in observation group after treatment was significantly higher than that in control group(t=6.884, P<0.05)and the PI was significantly lower than that in control group (t=4.979, P<0.05).Conclusion:Butylphthalide Injection can effectively correct the abnormality of brain hemodynamics in the ACI patients, enhance the ability of body in maintaining the stability of cerebral vascular perfusion, improve the neurological symptoms in the patients with ACI, and improve the therapeutic effects.
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[Abstract ] Objective The purpose of this study was to observe the effects of dl-3n-butylphthalide (NBP) sodium chloride injection post-processing on the expressions of X-inhibitor of apoptosis (XIAP) and Bcl-2/adenovirus E1B19kDa interacting protein 3 (BNIP3) in the hippocampus CA1 neurons of focal cerebral ischemia reperfusion (IR) rats, and to investigate the brain-protection mechanisms of NBP. Methods A total of65 adult male Sprague-Dawley rats were divided into five groups of equal number, sham op-eration, IR, and low-,medium -and high-dose NBP, according to the random number table. The IR models were established by modified ligation of the middle cerebral artery.The animals in the NBP groups received intra-abdominal injection of NBP at 2, 4, and 6 mg/kg, re-spectively.All the rats were sacrificed at 24 hours after modeling,neurological scores obtained by Zea Longa, the volume of infarction measured by TTC staining, the number of apoptotic cells counted by TUNEL, and the expressions of XIAP and BNIP3 detected by immunohistochemistry and real-time PCR. Results The neural function defect scores were markedly lower in low-, medium-and high-dose NBP groups than in IR model rats (P<0.05), with statis-tically significant differences among the three dose groups (P<0.05).The volume of infarction was remarkably higher in the low-dose than in the medium-and high-dose NBP groups (P<0.05).The number of apoptotic cells in the hippocampus CA1 neurons was de-creased in the NBP groups as compared with the IR models (P<0.05).The XIAP-and BNIP3-positive cells were significantly in-creased in the IR model rats as compared with the sham operation group ([22.31 ±0.94] and [60.13 ±2.59]/HP vs [3.07 ±1.43] and [5.78 ±0.44]/HP, P<0.05).In comparison with the IR models, the NBP-treated rats showed a progressively increased number of XIAP-positive cells in low-, medium-, and high-dose groups ([28.70 ±1.18], [32.79 ±0.88], and [37.01 ±1.24]/HP) (P<0.05) but a decreased number of BNIP3-positive cells in the three dose groups ([52.07 ±1.02], [40.30 ±2.00], and [31.04 ± 0.43]/HP) (P<0.05).Similarly, the expression of XIAP mRNA was up-regulated while that of BNIP3 mRNA down-regulated in the NBP treatment groups as compared with the IR model rats, both in a dose-dependent manner (P<0.05). Conclusion NBP post-processing has a neuroprotective effect on IR rats, which is associated with its impact on the expressions of XIAP and BNIP3.
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Objective To observe the effects of Dl-3-n-butylphthalide ( NBP) on the mitochondria infarction, size of myocardial infarction and myocardial apoptosis after acute myocardial ischemia in rats. Methods 92 male SD rats were divided into sham operation group (8 rats) , model group (21 rats) , and low-dose NBP group (21 rats) , medium-dose NBP group (21 rats) , high-dose NBP group (21 rats) . The model and NBP groups were made into MI model by ligation of the left anterior descending ( LAD) coronary artery, but not in sham-operated group. Model group and NBP group were taken heart specimens after coronary artery ligation. Cardiomyocyte apoptosis was ana-lyzed by TUNEL in each group. Size of MI was analyzed by TTC staining in sham-operated group, model group and high-dose NBP group. Electron perspective microscopy was applicated in observing mitochondria infarction in model group and high-dose NBP group after myocardial infarction. The expressions of Bcl-2 protein and Bax protein were detected by Western blot. Results Compared with model group, butylphthalide significantly increased expression of Bcl-2 protein ( P <0.05 ) and the ratio of Bcl-2/Bax ( P <0.05 ) , inhibited mitochondria infarction ( P <0.05 ) , reduced myocardial infarct size ( P<0.01 ) and cardiomyocyte apoptosis ( P<0.05 ) . Conclusion Bu-tylphthalide significantly inhibits myocardial infarction by increasing expression of Bcl-2 protein and the ratio of Bcl-2/Bax and decreasing mitochondria infarction, reducing myocardial infarct size and cardiomyocyte apoptosis in rats during the acute myocardial ischemia process.
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Objective To investigate the protective effect and its mechanism of DL-3-n-Butylphthalide on the brain damage in rats following whole brain irradiation.Methods A total of 120 male Sprague Dawley rats were randomly divided into sham-irradiation group,irradiatien group and DL-3-n-Butylphthalide group.The model of whole-brain irradiatien was established by exposuring rat brain to 4 MeV X-rays with a single-dose of 10 Gy.The rats were intraperitoneally injected with DL-3-n-Butylphthalide at the dosages of 0.3,1.0,and 3.0 mg/kg once a day.The contents of malondialdchyde and super oxide dismutase activity were measured,while the expressions of apoptosis-associated genes and the ultrastructural changes in hippocampus were examined by immunohistnchemisty staining and electron microscope,respectively.Results After irradiation,the content of malondialdehyde and the expression of apoptosis gene bax in rat brain tissue increased while the activity of super oxide dismutase(SOD) and the expression of anti-apoptosis gene bcl-2 decreased.Apoptosis was also observed in the neurons of hippocampus CA1.Compared with irradiation group,the content of malondialdehyde and the expression of bax gene in the DL-3-n-Butylphthalide group wen significantly reduced ( t =-3.89--1.96,2.72-3.48,P ＜ 0.05 ),while the activity of SOD and bcl-2 gene were significantly elevated ( t =2.94-3.76,-3.18--2.08,P ＜ 0.05),and the injury degree of neuron structure in the DL-3-n-Butylphthalide group was slighter than that in the irradiation group.Conclusions DL-3-n-Butylphthalide executes protective effects in a dose-dependent manner againest the radiation injury in rats brain by reducing the induction of malondialdehyde,raising the activity of SOD and inhibiting the generation of apoptosis.
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Objective To explore the effects of dl-3-n-butylphthalide on learning-memory ability and Na+-K+-ATPase activity of mitochondria in rats with chronic cerebral hypoperfusion.Methods Sprague-Dawlay rats were randomly divided into sham group( n= 10) ,model group (Permanent occlusion of bilateral common cawater maze test.Na+-K+-ATPase activity in mitochondria of forebrain tissue were also measured.Results Compared with sham group, memory ability of rats in model group decreased obviously( ( 10.41 ± 3.81 ), ( 25.54 prot- 1 , p ＜0.05 ).The memory ability of rats in large/small dose dl-3-n-butylphthalide groups both increased notably compared with the model group( respectively( 11.72 ± 5.78 ) s and ( 12.48 ± 5.45 ) s, P ＜ 0.01 ) and Na + -0.05 ).The effectives of large dose dl-3-n-butylphthalide and that of small dose dl-3-n-butylphthalide have no significantly differences.Conclusion Dl-3-n-butylphthalide could increase the Na + -K +-ATPase activity and protect mitochondria, improve the learning-memory ability of rats with chronic cerebral hypoperfusion.
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@#dl-3-n-Butylphthalide can be applied in many areas of central nervous system diseases,such as cerebral ischemia,cerebral trauma,dysmnesia,convulsion,and so on.This paper reviewed the effect and mechanism of it.
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AIM To study the effect of dl-3-n-butylphthalide (dl-NBP) on arachidonic acid(AA) release and phospholipase A2 (PLA2) mRNA in cerebral cortex of rats subjected to focal cerebral ischemia. METHODS Focal cerebral ischemia was induced by inserting a monofilament nylon suture into the internal carotid artery and blocking the origin of the middle cerebral artery. AA was determined with high performance liquid chromatography (HPLC). The PLA2 mRNA expression was evaluated by Northern blot analysis. RESULTS Six hours of cerebral ischemia induced AA release in the ischemic cerebral cortex. dl-NBP (10 or 20 mg·kg-1) and nimodipine (0.5 mg·kg-1) given intraperitoneally 5 min and 120 min again after the onset of ischemia significantly reduced AA concentration in the cerebral cortex (P<0.01). d-NBP, but not l-NBP, decreased AA release in the brain after middle cerebral artery occlusion. The expression of PLA2 mRNA in cerebral cortex induced by cerebral ischemia was also inhibited by dl-NBP and d-NBP (10 or 20 mg·kg-1, ip). CONCLUSION dl-NBP and d-NBP inhibited AA release and PLA2 mRNA expression in the ischemic brain tissue in vivo.