picroside-ii and Brain-Ischemia

Stroke is a common neurodegenerative disease in the wide world, and mitochondrial defects underlie the pathogenesis of ischemia, especially during reperfusion. Picroside II, the principal active component of Picrorhiza, is a traditional Chinese medicine. Our previous study demonstrated that the best therapeutic dose and time window were injection of picroside II at a dose of 10-20 mg/kg body weight following cerebral ischemia by 1.5-2.0 h. In this paper, the neuroprotective effect and the mechanism of picroside II were investigated, as well as its involvement in antioxidant and mitochondria cytochrome C (CytC) signal pathway following ischemia reperfusion (I/R) injury in rats. After 24 h of cerebral I/R, the neurobehavioral function was measured by modified neurological severity score test; the content of reactive oxygen species in brain tissue was measured by enzyme-linked immunosorbent assay; the cerebral infarction volume was detected by TTC staining; the morphology of brain tissue was observed by hematoxylin-eosin; the apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay; the ultrastructure of the cortical brain tissues was observation by transmission electron microscopy; the expressions of CytC and Caspase-3 were determined by immunohistochemical assay and Western blot. The results indicated that picroside II could scavenge ROS contents, decrease the cerebral infarction volume and apoptotic cells, protect the structure of mitochondria, down-regulate the expression of CytC and Caspase-3 in cerebral I/R rats. It can be concluded that picroside II exerts a neuroprotective effect by inhibiting the mitochondria CytC signal pathway following ischemia reperfusion injury in rats.

Topics: Animals; Antioxidants; Apoptosis; Brain; Brain Ischemia; Cinnamates; Cytochrome c Group; Iridoid Glucosides; Male; Mitochondria; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction

To explore the neuroprotective effect and mechanism of picroside II on extracellular regulated protein kinases1/2 (ERK1/2) signal transduction pathway in cerebral ischemia injuryrats. METHODS The middle cerebral artery occlusion (MCAO) model was established by inserting a monofilament into middle cerebral artery. Totally 96 successfully modeled Wistar rats were divided into the modelgroup, the treatment (picroside II) group, the Lipopolysachcaride (LPS) group, and the U0126 group according to random digit table. Each group was further divided into 3 subgroups, i.e. 6, 12, and 24 h sub-groups. Picroside II (20 mg/kg) was peritoneally injected to rats in the treatment group 2 h after ischemia.LPS (20 mg/kg) and Picroside II (20 mg/kg) were peritoneally injected to rats in the LPS group 2 h after ischemia. U0126-EtOH (20 mg/kg)and Picroside II (20 mg/kg) were peritoneally injected to rats in the U0126group 2 h after ischemia. Equal volume of normal saline was peritoneally injected to rats in the control groupand the model group. The neurobehavioral function was evaluated by modified neurological severity score(mNSS) test. The structure of neurons was observed using hematoxylin-eosinstaining (HE) staining. Theapoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of phosphorylated extracellular signal-regulated protein kinase1,2 (pERK1,2) in cortex was detected using immunohistochemistry (IHC) and Western blot.. After cerebral ischemia injury neurological impairment score increased, the damage of neuron in the cortical area was aggravated, apoptotic cells increased in the model group as time went by. The expression of pERK1/2 increased more significantly in the model group than in the control group (P <0.05). The damage of neuron in the cortical area was milder, while apoptotic cells decreased, the expression of pERK1f2 obviously decreased more in the treatment group and the U0126 group (P < 0.05). The early damage of neuron in the cortical area was more severe, apoptotic cells and the expression of pERK12 were comparatively higher in early stage of the LPS group, but the expression of pERK1/2 was somewhat decreased in late stage.. Activating ERK12 pathway could mediate apoptosis and inflammatory reactions of neurons after cerebral ischemia injury. Picroside II could protect the nerve system possibly through reducing activation of ERKI2 pathway, inhibiting apoptosis of neurons and inflammation reaction.

Topics: Animals; Apoptosis; Brain Ischemia; Cinnamates; Infarction, Middle Cerebral Artery; Iridoid Glucosides; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; Random Allocation; Rats; Rats, Wistar

This paper aimed to explore the protective effects of picroside II against the neuronal apoptosis and changes in morphology and structure that follow cerebral ischemic injury in rats. A focal cerebral ischemic model was established by inserting a monofilament thread to achieve middle cerebral artery occlusion (MCAO) in 60 Wistar rats, and intraperitoneal injections of picroside II (20 mg/kg) were administered. The neurobehavioral functions were evaluated with the modified neurological severity score (mNSS) test. The cerebral infarct volumes were measured with tetrazolium chloride (TTC) staining. The morphology and ultrastructure of the cortical brain tissues were observed with hematoxylin-eosin staining and transmission electron microscopy, respectively. The apoptotic cells were counted with terminal deoxynucleotidyl transferase dUTP nick-end labeling and flow cytometry, and pERK1/2 expression was determined by immunohistochemical assay and Western blot. The results indicated that neurological behavioral malfunctions and cerebral infarcts were present in the MCAO rats. In the model group, the damage to the structures of the neurons and the blood brain barrier (BBB) in the cortex was more severe, and the numbers of apoptotic cells, the early apoptotic ratio (EAR) and pERK1/2 expression were significantly increased in this group compared to the control group (P<0.05). In the treatment group, the neurological behavioral function and the morphology and ultrastructure of the neurons and the BBB were improved including the number of Mi increased and relative area of condensed chromosome and basement (BM) thickness descreased, and the cerebral infarct volume, the number of apoptotic cells, the EAR and pERK1/2 expression were significantly decreased compared to the model group (P<0.05). These results suggest that picroside II reduced apoptosis and improved the morphology and ultrastructure of the neurons and the BBB and that these effects resulted in the recovery of the neurobehavioral function of rats with cerebral ischemia.

Topics: Animals; Apoptosis; Blotting, Western; Brain; Brain Ischemia; Cinnamates; Flow Cytometry; Immunohistochemistry; In Situ Nick-End Labeling; Iridoid Glucosides; Male; Microscopy, Electron, Transmission; Neurons; Rats; Rats, Wistar

To explore the neuroprotective effect and optimize the therapeutic dose and time window of picroside II by orthogonal test and the expression of myelin basic protein (MBP) in cerebral ischemic injury in rats. Bilateral common carotid artery occlusion (BCCAO) was used to establish forebrain ischemia models. The successful rat models were grouped according to orthogonal experimental design and injected picroside II intraperitoneally at different ischemic time with different doses. Myelin sheath fast green staining(FGS) and transmission electron microscopy (TEM) were used to observe nerve fiber myelin; the expression of MBP was tested qualitatively and quantitatively by immunohistochemical assay (IHC) and Western blot (WB); Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transcription level of MBP mRNA.. The protective effect of picroside II was presented by increasing the expression of MBP and decreasing demyelination after cerebral ischemic injury. The best therapeutic time window and dose was (1) ischemia 2.0 h with picroside II 10 mg/kg body weight according to the results of FGS, IHC and WB; (2) ischemia 1.5 h with picroside II 20 mg/kg according to the analysis of RT-PCR.. Given the principle of the longest time window and the lowest therapeutic dose, the optimized therapeutic dose and time window should be injecting picroside II intraperitoneally with 10-20 mg/kg body weight at ischemia 1.5-2.0 h in cerebral ischemic injury.

Topics: Animals; Brain Ischemia; Cinnamates; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation; Injections, Intraperitoneal; Iridoid Glucosides; Male; Myelin Basic Protein; Myelin Sheath; Neuroprotective Agents; Rats; Rats, Wistar; Treatment Outcome

The aim of this study was to explore the optimal therapeutic dose and time window of picroside II for treating cerebral ischemic injury in rats according to the orthogonal test. The middle cerebral artery occlusion (MCAO) models were established by intraluminally inserting a thread into middle cerebral artery (MCA) from left external carotid artery (ECA). The successful rat models were randomly divided into 16 groups according to the orthogonal layout of [L(16)(4(5))] and treated by injecting picroside II intraperitoneally with different doses at various times. The neurological behavioral function was evaluated by Bederson's test and the cerebral infarction volume was measured by tetrazolium chloride (TTC) staining. The expressions of neuron specific enolase (NSE) and neuroglial mark-protein S-100 were determined by immunohistochemisty assay. The results indicated that the optimal compositions of the therapeutic dose and time window of picroside II in treating cerebral ischemic injury were ischemia 1.5 h with 20 mg/kg body weight according to Bederson's test, 1.0 h with 20 mg/kg body weight according to cerebral infarction volume, 1.5 h with 20 mg/kg body weight according to the expressions of NSE and S-100 respectively. Based on the principle of the minimization of medication dose and maximization of therapeutic time window, the optimal composition of the therapeutic dose and time window of picroside II in treating cerebral ischemic injury should be achieved by injecting picroside II intraperitoneally with 20 mg/kg body weight at ischemia 1.5 h.

Topics: Analysis of Variance; Animals; Brain Ischemia; Cinnamates; Dose-Response Relationship, Drug; Iridoid Glucosides; Male; Phosphopyruvate Hydratase; Rats, Wistar; S100 Proteins; Time Factors

To explore the effects of picrodide II on the expressions of Toll-like receptor 4 (TLR4) and nuclear transcription factor kappaB (NFkappaB) in brain tissue of rat after cerebral ischemic reperfusion (I/R) injury.. Ten rats from 60 adult healthy female Wistar rats received sham-operation were set as the sham-operative group. Established as middle cerebral I/R model (MCAO/R) by thread tying method, the 30 successfully modeled rats were equally randomized into the negative control group, the positive control group and the treatment group. Besides, rats in the treatment group and the positive control group were respectively intervened with picrodide II (10 mg/kg) and salvianic acid A sodium (10 mg/kg) via caudal vein injection before I/R injury, while rats in the sham-operative group and the negative group were injected with equal volume of 0.1 mol/L PBS. Immunohistochemistry stain was used to determine the expressions of TLR4 and NFkappaB, and the apoptotic cells were counted by TUNEL-immunofluorescence assay.. In the sham-operative group, the TLR4 and NFkappaB expressed weakly with few TUNEL positive cells scattering in the cortex, striatum and hippocampus. As compared with the sham-operative group, TLR4 and NFkappaB in the negative control group were significantly higher both in absorption A) value and cell number (P < 0.05). In the treatment group and the positive control group, the expressions of TLR4 and NFkappaB and the number of TUNEL positive cells were significantly lower than those in the negative control group (P < 0.05), but no significant difference was shown between the two treated groups (P > 0.05).. Picroside II could down-regulate the expressions of TLR4 and NFkappaB, and inhibit the inflammatory response induced apoptosis in cerebral I/R injured rats.

Topics: Animals; Apoptosis; Brain Ischemia; Cinnamates; Disease Models, Animal; Female; Iridoid Glucosides; NF-kappa B; Rats; Rats, Wistar; Reperfusion Injury; Toll-Like Receptor 4

Excitatory amino acid toxicity, oxidative stress, intracellular calcium overload, as well as inflammation and apoptosis are involved in the pathological process after cerebral ischemic reperfusion injury. Picrodide 2 could inhibit neuronal apoptosis and play anti-oxidant and anti-inflammation role in cerebral ischemia/reperfusion injuries, but the exact mechanism is not very clear. This study aims to explore the anti-inflammation mechanism of picroside 2 in cerebral ischemic reperfusion injury in rats.. The middle cerebral artery occlusion reperfusion models were established with intraluminal thread methods in 90 adult healthy female Wistar rats. Picroside 2 and salvianic acid A sodium were respectively injected from tail vein at the dosage of 10 mg/kg for treatment. The neurobehavioral function was evaluated with Bederson's test and the cerebral infarction volume was observed with tetrazolium chloride (TTC) staining. The apoptotic cells were counted by in situ terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The immunohistochemistry stain was used to determine the expressions of toll-like receptor 4 (TLR4), nuclear transcription factor kappaB (NFkappaB) and tumor necrosis factor alpha (TNFalpha). The concentrations of TLR4, NFkappaB and TNFalpha in brain tissue were determined by enzyme linked immunosorbent assay (ELISA).. After cerebral ischemic reperfusion, the rats showed neurobehavioral function deficit and cerebral infarction in the ischemic hemisphere. The number of apoptotic cells, the expressions and the concentrations in brain tissue of TLR4, NFkappaB and TNFalpha in ischemia control group increased significantly than those in the sham operative group (P < 0.01). Compared with the ischemia control group, the neurobehavioral scores, the infarction volumes, the apoptotic cells, the expressions and concentrations in brain tissue of TLR4, NFkappaB and TNFalpha were obviously decreased both in the picroside 2 and salvianic acid A sodium groups (P < 0.01). There was no statistical difference between the two treatment groups in above indexes (P > 0.05).. Picroside 2 could down-regulate the expressions of TLR4, NFkappaB and TNFalpha to inhibit apoptosis and inflammation induced by cerebral ischemic reperfusion injury and improve the neurobehavioral function of rats.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Brain; Brain Ischemia; Caffeic Acids; Cinnamates; Disease Models, Animal; Encephalitis; Female; Glucosides; Infarction, Middle Cerebral Artery; Iridoid Glucosides; Lactates; Neuropsychological Tests; NF-kappa B; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Toll-Like Receptor 4; Treatment Outcome; Tumor Necrosis Factor-alpha