PHPB ameliorates memory deficits and reduces oxidative injury in Alzheimer's disease mouse model by activating Nrf2 signaling pathway.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common cause of dementia in elderly people and substantially affects patient quality of life. Oxidative stress is considered a key factor in the development of AD. Nrf2 plays a vital role in maintaining redox homeostasis and regulating neuroinflammatory responses in AD. Previous studies show that potassium 2-(1-hydroxypentyl)-benzoate (PHPB) exerts neuroprotective effects against cognitive impairment in a variety of dementia animal models such as APP/PS1 transgenic mice. In this study we investigated whether PHPB ameriorated the progression of AD by reducing oxidative stress (OS) damage. Both 5- and 13-month-old APP/PS1 mice were administered PHPB (100 mg·kg-1·d-1, i.g.) for 10 weeks. After the cognition assessment, the mice were euthanized, and the left hemisphere of the brain was harvested for analyses. We showed that 5-month-old APP/PS1 mice already exhibited impaired performance in the step-down test, and knockdown of Nrf2 gene only slightly increased the impairment, while knockdown of Nrf2 gene in 13-month-old APP/PS1 mice resulted in greatly worse performance. PHPB administration significantly ameliorated the cognition impairments and enhanced antioxidative capacity in APP/PS1 mice. In addition, PHPB administration significantly increased the p-AKT/AKT and p-GSK3ß/GSK3ß ratios and the expression levels of Nrf2, HO-1 and NQO-1 in APP/PS1 mice, but these changes were abolished by knockdown of Nrf2 gene. In SK-N-SH APPwt cells and primary mouse neurons, PHPB (10 µM) significantly increased the p-AKT/AKT and p-GSK3ß/GSK3ß ratios and the level of Nrf2, which were blocked by knockdown of Nrf2 gene. In summary, this study demonstrates that PHPB exerts a protective effect via the Akt/GSK3ß/Nrf2 pathway and it might be a promising neuroprotective agent for the treatment of AD.

PHPB Attenuated Cognitive Impairment in Type 2 Diabetic KK-Ay Mice by Modulating SIRT1/Insulin Signaling Pathway and Inhibiting Generation of AGEs.

Diabetes mellitus (DM) has been recognized as an increased risk factor for cognitive impairment, known as diabetic encephalopathy (DE). Hyperglycemia and insulin resistance are the main initiators of DE, which is related to the accumulation of advanced glycation end products (AGEs). Potassium 2-(1-hydroxypentyl)-benzoate (PHPB), a derivative of 3-n-butylphthalide (dl-NBP), has emerged various properties including improved mitochondrial function, antioxidant, anti-neuroinflammation, and neuroprotective effects. The present study aimed to investigate the neuroprotective effect of PHPB against AGEs accumulation in type 2 diabetic KK-Ay mice model with DE and further explore the underlying mechanisms. The results showed that PHPB markedly ameliorated the spatial learning ability of KK-Ay mice in the Morris water maze and decreased AD-like pathologic changes (Tau hyperphosphorylation) in the cortex. Furthermore, we found that PHPB treatment significantly reduced AGEs generation via up-regulation of glyoxalase-1 (GLO1) protein and enhancement of methylglyoxal (MG) trapping, while there was no obvious difference in levels of glucose in plasma or brain, contents of total cholesterol (TC), triglycerides (TG), and plasma insulin. Also, PHPB treatment improved the insulin signaling pathway by increasing sirtuin1 (SIRT1) deacetylase activity and attenuated oxidative stress evidenced by elevating glucose-6-phosphate dehydrogenase (G-6-PD) protein expression, promoting the production of reduced glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), restoring mitochondrial membrane potential, increasing adenosine triphosphate (ATP) generation, and reducing malondialdehyde (MDA) levels in the brain. Taken together, PHPB exhibited a beneficial effect on DE, which involved modulating the SIRT1/insulin signaling pathway and reducing oxidative stress by inhibiting the generation of AGEs.

A blood-brain barrier-penetrating AAV2 mutant created by a brain microvasculature endothelial cell-targeted AAV2 variant.

Upon systemic administration, adeno-associated virus serotype 9 (AAV9) and the capsid variant PHP.eB show distinct tropism for the central nervous system (CNS), whereas AAV2 and the capsid variant BR1 transduce brain microvascular endothelial cells (BMVECs) with little transcytosis. Here, we show that a single amino acid substitution (from Q to N) in the BR1 capsid at position 587 (designated BR1N) confers a significantly higher blood-brain barrier (BBB) penetration capacity to BR1. Intravenously infused BR1N showed significantly higher CNS tropism than BR1 and AAV9. BR1 and BR1N likely use the same receptor for entry into BMVECs; however, the single amino acid substitution has profound consequences on tropism. This suggests that receptor binding alone does not determine the final outcome in vivo and that further improvements of capsids within predetermined receptor usage are feasible.

Optimized AAV Vectors for TMC1 Gene Therapy in a Humanized Mouse Model of DFNB7/11.

Gene therapy for genetic hearing loss is an emerging therapeutic modality for hearing restoration. However, the approach has not yet been translated into clinical application. To further develop inner-ear gene therapy, we engineered a novel mouse model bearing a human mutation in the transmembrane channel-1 gene (Tmc1) and characterized the auditory phenotype of the mice. TMC1 forms the mechanosensory transduction channel in mice and humans and is necessary for auditory function. We found that mice harboring the equivalent of the human p.N199I mutation (p.N193I) had profound congenital hearing loss due to loss of hair cell sensory transduction. Next, we optimized and screened viral payloads packaged into AAV9-PHP.B capsids. The vectors were injected into the inner ears of Tmc1Δ/Δ mice and the new humanized Tmc1-p.N193I mouse model. Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), cell survival, and biodistribution were evaluated in the injected mice. We found broad-spectrum, durable recovery of auditory function in Tmc1-p.N193I mice injected with AAV9-PHP.B-CB6-hTMC1-WPRE. ABR and DPOAE thresholds were equivalent to those of wild-type mice across the entire frequency range. Biodistribution analysis revealed viral DNA/RNA in the contralateral ear, brain, and liver but no overt toxicity. We conclude that the AAV9-PHP.B-CB6-hTMC1-WPRE construct may be suitable for further development as a gene therapy reagent for treatment of humans with genetic hearing loss due to recessive TMC1 mutations.

Efficient Viral Transduction in Fetal and Adult Human Inner Ear Explants with AAV9-PHP.B Vectors.

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application.

The attenuation effect of potassium 2-(1-hydroxypentyl)-benzoate in a mouse model of diabetes-associated cognitive decline: The protein expression in the brain.

AIMS: dl-PHPB (potassium 2-(1-hydroxypentyl)-benzoate) has been shown to have neuroprotective effects against acute cerebral ischemia, vascular dementia, and Alzheimer's disease. The aim of this study was to investigate the effects of dl-PHPB on memory deficits and preliminarily explore the underlying molecular mechanism. METHODS: Blood glucose and behavioral performance were evaluated in the KK-Ay diabetic mouse model before and after dl-PHPB administration. Two-dimensional difference gel electrophoresis (2D-DIGE)-based proteomics was used to identify differentially expressed proteins in brain tissue. Western blotting was used to study the molecular mechanism of the related signaling pathways. RESULTS: Three-month-old KK-Ay mice were given 150 mg/kg dl-PHPB by oral gavage for 2 months, which produced no effect on the level of serum glucose. In the Morris water maze test, KK-Ay mice treated with dl-PHPB showed significant improvements in spatial learning and memory deficits compared with vehicle-treated KK-Ay mice. Additionally, we performed 2D-DIGE to compare brain proteomes of 5-month KK-Ay mice treated with and without dl-PHPB. We found 14 altered proteins in the cortex and 11 in the hippocampus; two of the 25 altered proteins and another four proteins that were identified in a previous study on KK-Ay mice were then validated by western blot to further confirm whether dl-PHPB can reverse the expression levels of these proteins. The phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase-3ß (PI3K/Akt/GSK-3ß) signaling pathway was also changed in KK-Ay mice and dl-PHPB treatment could reverse it. CONCLUSIONS: These results indicate that dl-PHPB may play a potential role in diabetes-associated cognitive impairment through PI3K/Akt/GSK-3ß signaling pathway and the differentially expressed proteins may become putative therapeutic targets.

Context-Specific Function of the Engineered Peptide Domain of PHP.B.

One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.

PhP.B Enhanced Adeno-Associated Virus Mediated-Expression Following Systemic Delivery or Direct Brain Administration.

Of the adeno-associated viruses (AAVs), AAV9 is known for its capability to cross the blood-brain barrier (BBB) and can, therefore, be used as a noninvasive method to target the central nervous system. Furthermore, the addition of the peptide PhP.B to AAV9 increases its transduction across the BBB by 40-fold. Another neurotropic serotype, AAV5, has been shown as a gene therapeutic delivery vehicle to ameliorate several neurodegenerative diseases in preclinical models, but its administration requires invasive surgery. In this study, AAV9-PhP.B and AAV5-PhP.B were designed and produced in an insect cell-based system. To AAV9, the PhP.B peptide TLAVPFK was added, whereas in AAV5-PhP.B (AQTLAVPFKAQAQ), with AQ-AQAQ sequences used to swap with the corresponding sequence of AAV5. The addition of PhP.B to AAV5 did not affect its capacity to cross the mouse BBB, while increased transduction of liver tissue was observed. Then, intravenous (IV) and intrastriatal (IStr) delivery of AAV9-PhP.B and AAV5 were compared. For AAV9-PhP.B, similar transduction and expression levels were achieved in the striatum and cortex, irrespective of the delivery method used. IStr administration of AAV5 resulted in significantly higher amounts of vector DNA and therapeutic miRNA in the target regions such as striatum and cortex when compared with an IV administration of AAV9-PhP.B. These results illustrate the challenge in developing a vector that can be delivered noninvasively while achieving a transduction level similar to that of direct administration of AAV5. Thus, for therapeutic miRNA delivery with high local expression requirements, intraparenchymal delivery of AAV5 is preferred, whereas a humanized AAV9-PhP.B may be useful when widespread brain (and peripheral) transduction is needed.

A Single Intravenous Injection of AAV-PHP.B-hNDUFS4 Ameliorates the Phenotype of Ndufs4 -/- Mice.

Leigh syndrome, or infantile necrotizing subacute encephalopathy (OMIM #256000), is one of the most common manifestations of mitochondrial dysfunction, due to mutations in more than 75 genes, with mutations in respiratory complex I subunits being the most common cause. In the present study, we used the recently described PHP.B serotype, characterized by efficient capacity to cross the blood-brain barrier, to express the hNDUFS4 gene in the Ndufs4 -/- mouse model of Leigh disease. A single intravenous injection of PHP.B-hNDUFS4 in adult Ndufs4 -/- mice led to a normalization of the body weight, marked amelioration of the rotarod performance, delayed onset of neurodegeneration, and prolongation of the lifespan up to 1 year of age. hNDUFS4 protein was expressed in virtually all brain regions, leading to a partial recovery of complex I activity. Our findings strongly support the feasibility and effectiveness of adeno-associated viral vector (AAV)-mediated gene therapy for mitochondrial disease, particularly with new serotypes showing increased permeability to the blood-brain barrier in order to achieve widespread expression in the central nervous system.

Studies of pathology and pharmacology of diabetic encephalopathy with KK-Ay mouse model.

AIMS: Pathogenesis of diabetic encephalopathy (DE) is not completely understood until now. The purposes of this study were to illustrate the changes in morphology, function, and important transporters in neurons and glia during DE, as well as to reveal the potential therapeutic effects of medicines and the diet control on DE. METHODS: Spontaneous obese KK-Ay mice were used to investigate diabetes-induced cognitive disorder, the morphology, function, and protein expression changes in impact animal and the cell level studies. The new drug candidate PHPB, donepezil, and low-fat food were used to observe the therapeutic effects. RESULTS: KK-Ay mice at 5 months of age showed typical characteristics of type 2 diabetes mellitus (T2DM） and appeared significant cognitive deficits. Morphological study showed microtubule-associated protein 2 (MAP2) expression was increased in hippocampal neurons and glial fibrillary acidic protein (GFAP) expression decreased in astrocytes. Meanwhile, the vesicular glutamate transporter 1 (vGLUT1) expression was increased and glucose transporter 1 (GLUT1) decreased, and the expression of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was also reduced in KK-Ay mice. Microglia were activated, and IL-1ß and TNF-α were increased obviously in the brains of the KK-Ay mice. Most of the above changes in the KK-Ay mice at 5 months of age could be relieved by diet intervention (DR) or by treatment of donepezil or new drug candidate PHPB. CONCLUSION: KK-Ay mouse is a useful animal model for studying DE. The alterations of morphology, structure, and function of astrocyte and microglia in KK-Ay mice might be rescued by DR and by treatment of medicine. The proteins we reported in this study could be used as biomarkers and the potential drug targets for DE study and treatment.

Potassium 2-(1-hydroxypentyl)-benzoate improves depressive-like behaviors in rat model.

Potassium 2-(1-hydroxypentyl)-benzoate (PHPB) is a novel drug candidate for acute ischemic stroke. PHPB has been also shown to be beneficial for some neurodegenerative diseases. In this study, we demonstrated that PHPB improved depressive-like behaviors induced by chronic unpredictable mild stress (CUMS) in rats. Male SD rats were subjected to the stress for five weeks. PHPB (30 and 100 mg/kg) or fluoxetine (FLX 10 mg/kg, as positive control) was administered orally from the third week in CUMS procedure. The behavioral tests were applied and then the biochemical studies were carried out. PHPB or FLX treatment rescued the behavioral deficiency in CUMS-exposed rats. Meanwhile, PHPB normalized the enhanced level of serum corticosterone, improved hippocampal and serum BDNF levels, as well as p-CREB level in hippocampus. In addition, PHPB could reverse the reduced level of extracellular 5-HT and its metabolite 5-HIAA in prefrontal cortex (PFC) of depressed rats. In summary, our results showed that PHPB improved depression-like behaviors in CUMS-exposed rats. The mechanisms might relate to the reverse of neurotrophic disturbance in the brain, reducing excessive HPA axis response and facilitating the release of 5-HT.

Potassium 2-(1-hydroxypentyl)-benzoate improves depressive-like behaviors in rat model

Potassium 2-(1-hydroxypentyl)-benzoate (PHPB) is a novel drug candidate for acute ischemic stroke. PHPB has been also shown to be beneficial for some neurodegenerative diseases. In this study, we demonstrated that PHPB improved depressive-like behaviors induced by chronic unpredictable mild stress (CUMS) in rats. Male SD rats were subjected to the stress for five weeks. PHPB (30 and 100 mg/kg) or fluoxetine (FLX 10 mg/kg, as positive control) was administered orally from the third week in CUMS procedure. The behavioral tests were applied and then the biochemical studies were carried out. PHPB or FLX treatment rescued the behavioral deficiency in CUMS-exposed rats. Meanwhile, PHPB normalized the enhanced level of serum corticosterone, improved hippocampal and serum BDNF levels, as well as p-CREB level in hippocampus. In addition, PHPB could reverse the reduced level of extracellular 5-HT and its metabolite 5-HIAA in prefrontal cortex (PFC) of depressed rats. In summary, our results showed that PHPB improved depression-like behaviors in CUMS-exposed rats. The mechanisms might relate to the reverse of neurotrophic disturbance in the brain, reducing excessive HPA axis response and facilitating the release of 5-HT.

Potassium 2-(1-hydroxypentyl)-benzoate attenuates neuronal apoptosis in neuron-astrocyte co-culture system through neurotrophy and neuroinflammation pathway.

Potassium 2-(1-hydroxypentyl)-benzoate (d,l-PHPB), a new drug candidate for ischemic stroke at the phase II clinic trial, has been shown to protect neurons by inhibiting oxidative injury and reducing neuron apoptosis in previous studies. But the mechanisms of d,l-PHPB remain to be studied. In this study, a neuron-astrocytes co-culture system was used to elucidate the roles of astrocytes in neuroprotection of d,l-PHPB under oxygen-glucose deprivation/reoxygenation (OGD/R) condition. Our data showed that d,l-PHPB reduced neuronal apoptosis in mono-culture system and this effect was enhanced in neuron-astrocyte co-culture system under the OGD/R condition. Meanwhile, d,l-PHPB obviously increased the levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which were mainly secreted from astrocytes, in the co-culture system after OGD/R. The PI3K/AKT and ERK signaling pathways as well as the p-TRKA/B receptors were involved in the process. In addition, the levels of TNF-α and IL-1ß secreted from astrocytes after OGD/R were markedly reduced after d,l-PHPB treatment, which was mainly due to the suppression of phosphorylated p38. In conclusion, the present study demonstrates that the neuroprotective effects of d,l-PHPB were improved by astrocytes, mainly mediated by increasing the release of BDNF/NGF and attenuating inflammatory cytokines.

Potassium 2-(l-hydroxypentyl)-benzoate attenuates neuroinflammatory responses and upregulates heme oxygenase-1 in systemic lipopolysaccharide-induced inflammation in mice.

A neuroinflammatory response is commonly involved in the progression of many neurodegenerative diseases. Potassium 2-(1-hydroxypentyl)-benzoate (PHPB), a novel neuroprotective compound, has shown promising effects in the treatment of ischemic stroke and Alzheimer׳s disease (AD). In the present study, the anti-inflammatory effects of PHPB were investigated in the plasma and brain of C57BL/6 mice administered a single intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Levels of iNOS and the cytokines TNFα, IL-1ß and IL-10 were elevated in plasma, cerebral cortex and hippocampus after LPS injection and the number of microglia and astrocytes in cortex and hippocampus were increased. LPS also upregulated the expression of heme oxygenase-1 (HO-1) in the cortex and hippocampus. PHPB reduced the levels of iNOS and cytokines in the plasma and brain, decreased the number of microglia and astrocytes and further enhanced the upregulation of HO-1. In addition, PHPB inhibited the LPS-induced phosphorylation of ERK, P38 and JNK. These results suggest that PHPB is a potential candidate in the treatment of neurodegenerative diseases through inhibiting neuroinflammation.

Potassium 2-(l-hydroxypentyl)-benzoate attenuates neuroinflammatory responses and upregulates heme oxygenase-1 in systemic lipopolysaccharide-induced inflammation in mice

A neuroinflammatory response is commonly involved in the progression of many neurodegenerative diseases. Potassium 2-(1-hydroxypentyl)-benzoate (PHPB), a novel neuroprotective compound, has shown promising effects in the treatment of ischemic stroke and Alzheimer׳s disease (AD). In the present study, the anti-inflammatory effects of PHPB were investigated in the plasma and brain of C57BL/6 mice administered a single intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Levels of iNOS and the cytokines TNF, IL-1and IL-10 were elevated in plasma, cerebral cortex and hippocampus after LPS injection and the number of microglia and astrocytes in cortex and hippocampus were increased. LPS also upregulated the expression of heme oxygenase-1 (HO-1) in the cortex and hippocampus. PHPB reduced the levels of iNOS and cytokines in the plasma and brain, decreased the number of microglia and astrocytes and further enhanced the upregulation of HO-1. In addition, PHPB inhibited the LPS-induced phosphorylation of ERK, P38 and JNK. These results suggest that PHPB is a potential candidate in the treatment of neurodegenerative diseases through inhibiting neuroinflammation.

Potassium 2-(1-hydroxypentyl)-benzoate attenuates neuronal apoptosis in neuron-astrocyte co-culture system through neurotrophy and neuroinflammation pathway

Potassium 2-(1-hydroxypentyl)-benzoate (d,l-PHPB), a new drug candidate for ischemic stroke at the phase II clinic trial, has been shown to protect neurons by inhibiting oxidative injury and reducing neuron apoptosis in previous studies. But the mechanisms of d,l-PHPB remain to be studied. In this study, a neuron-astrocytes co-culture system was used to elucidate the roles of astrocytes in neuroprotection of d,l-PHPB under oxygen-glucose deprivation/reoxygenation (OGD/R) condition. Our data showed that d,l-PHPB reduced neuronal apoptosis in mono-culture system and this effect was enhanced in neuron-astrocyte co-culture system under the OGD/R condition. Meanwhile, d,l-PHPB obviously increased the levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which were mainly secreted from astrocytes, in the co-culture system after OGD/R. The PI3K/AKT and ERK signaling pathways as well as the p-TRKA/B receptors were involved in the process. In addition, the levels of TNF-and IL-1secreted from astrocytes after OGD/R were markedly reduced after d,l-PHPB treatment, which was mainly due to the suppression of phosphorylated p38. In conclusion, the present study demonstrates that the neuroprotective effects of d,l-PHPB were improved by astrocytes, mainly mediated by increasing the release of BDNF/NGF and attenuating inflammatory cytokines.

Dl-PHPB inhibits platelet aggregation and affects the balance of PGI2/TXA2 and fibrinolytic system of rats / 中国药学杂志

OBJECTIVE: To investigate the effect of potassiam 2-(1-hydroxypentyl)-benzoae (dl/-PHPB) on platelet aggregation, the balance of PGI2/TXA, and fibrinolytic system of rats. METHODS: Rats were randomly divided into 5 groups; control group, low (1.3 mg·kg-1), middle (3.9 mg·kg-1) and high (12.9 mg·kg-1) dose of dl-PHPB groups and ticlopidine group. Blood was taken from the abdominal aorta 30 min after dl-PHPB administrated intravenously, and the rate of platelet aggregation was detected by platelet aggregometer. The expression of CD62p on the surface of platelet was determined by flow cytometry. The contents of 6-Keto-PGF1α, thromboxane B2 (TXB2), t-PA and PAT-1 in serum were measured by radio-immuno assay and FLISA kit, respectively. RESULTS: dl-PHPB inhibited rat platelet aggregation induced by A DP in dose-dependent manner more potently compared with AA, collagen and thrombin. dl-PHPB also decreased the expression of CD62p. It increased the level of 6-keto-PGF1α, the metabolite of prostaglandin I2 (PGL2) and reduced the content of PAI-1. But dl/-PHPB had no effect on the contents of TXB2 the metabolite of TXA2 and t-PA. CONCLUSION: dl-PHPB may significantly inhibit rat platelet aggregation induced by ADP, increase the ratio of PG12/TXA2 and enhance the activity of fibrinolytic system.