Picroside Ⅱ down-regulates matrix metalloproteinase-9 expression following cerebral ischemia/reperfusion injury in rats

Studies have shown that Picroside Ⅱ attenuates inflammatory reactions following brain ischemia through the inhibition of the TLR-4-NF-KB signal transduction pathway, and ameliorates cerebral edema through the reduction of aquaporin-4 expression. Matrix metalloproteinase-9 （MMP-9）, located downstream of the TLR-4-NF-KB signal transduction pathway, can degrade the neurovascular matrix, damage the blood-brain barrier to induce cerebral edema, and directly result in neuronal apoptosis and brain injury, Therefore, the present study further observed MMP-9 expression in the brain tissues of rats with cerebral ischemia/reperfusion injury following Picroside Ⅱ treatment. Results demonstrated that Picroside Ⅱ significantly reduced MMP-9 expression in ischemic brain tissues, as well as neuronal apoptosis and brain infarct volume, suggesting Picroside Ⅱ exhibits neuroprotection by down-regulating MMP-9 expression and inhibiting cell apoptosis.

Anti-oxidant effect of picroside II in a rat model of cerebral ischemia/reperfusion injury

Picroside II,the major active component of picroside,has been shown to induce PC12 cell axonal growth and relieve free radical damage.In vivo experiments have demonstrated that picroside II can improve neurological function in rats with cerebral ischemia/reperfusion injuries.In the present in vivo study,enzyme-linked immunosorbent assay and immunohistochemistry revealed that picroside II increased superoxide dismutase content and reduced inducible nitric oxide synthase content in the ischemic hemisphere.The effects of picroside II were similar to those of salvianic acid A sodium,an active control drug.These results indicate that picroside II exerts a neuroprotective effect,possibly by downregulating inducible nitric oxide synthase expression,increasing superoxide dismutase activity,and inhibiting neuronal apoptosis.

Protective mechanisms of picroside Ⅱ on aquaporin-4 expression in a rat model of cerebral ischemia/reperfusion injury

BACKGROUND： Aquaporin-4 （AQP-4） over-expression following cerebral ischemia results in cerebral edema. Picroside Ⅱ has been shown to exhibit a neuroprotective effect on neuronal apoptosis. However, few reports have addressed the neuroprotective mechanisms and therapeutic times following cerebral ischemic reperfusion injury. OBJECTIVE： To explore the neuroprotective effects and ideal treatment window for picroside Ⅱ treatment of middle cerebral artery occlusion and reperfusion injury in rats. DESIGN, TIME AND SETTING; A randomized, controlled, animal experiment was performed at Institute of Cerebrovascular Diseases, Qingdao University Medical College from September 2008 to May 2009. MATERIALS： Picroside II was purchased from Tianjin Kuiqing Medical Technology, China. METHODS： A total of 165 adult, healthy, male, Wistar rats were randomly assigned to sham-surgery （n = 15）, model （n = 75）, and treatment groups （n = 75）. Rats in the model and treatment groups underwent middle cerebral artery occlusion and reperfusion through the use of an intraluminal monofilament suture on the left external-internal carotid artery, The treatment group was injected with 1.0% picroside Ⅱ （10 mg/kg） into the tail vein, and the model and sham-surgery groups were injected with 0.1 mol/L phosphate buffered saline （250 μL）. MAIN OUTCOME MEASURES： Neurological functional scores were evaluated using the Longa＇s method; cerebral infarction volume was detected through the use of tetrazolium chlodde staining; cellular apoptosis was determined through the use of the in situ end-labeling method; aquaporin-4 expression was measured using fluorescence labeling analysis and reverse transcription polymerase chain reaction technique. RESULTS： At 0.5 hour following cerebral ischemic injury, neurological functional scores were low, and a small infarction focus was detected in the ischemic cortex of the model group. Along with prolonged ischemia and an increased number of apoptosis-positive cells, AQP-4 mRNA and protein expression was increased. At 1-2 hours after ischemia, neurological scores and infarction sizes were significantly increased in the model group. Apoptotic-positive cells were widespread in the ipsilateral cortex and stdatum. In addition, AQP-4 mRNA and protein expression levels were increased. Picroside II treatment significantly decreased neurological scores and infarction volume, and reduced AQP-4 mRNA and protein expression levels compared with the model group （P 〈 0.05 or P 〈 0.01）. At 1 hour after ischemia, the therapeutic effect of picroside Ⅱ was notable （P 〈 0.01）. CONCLUSION： Picroside Ⅱ played a protective role in cerebral ischemic reperfusion injury by inhibiting apoptosis and regulating AQP-4 expression. The best therapeutic time window was 1 hour after cerebral ischemic reperfusion.

Optimal therapeutic dose and time window of picroside II in cerebral ischemic injury

A preliminary study from our research group showed that picroside II inhibited neuronal apop- tosis in ischemic penumbra, reduced ischemic volume, and improved neurobehavioral function in rats with cerebral ischemia. The aim of the present study was to validate the neuroprotective effects of picroside II and optimize its therapeutic time window and dose in a rat model of cerebral ischemia. We found that picroside Ⅱ inhibited cell apoptosis and reduced the expression of neuron-specific enolase, a marker of neuronal damage, in rats after cerebral ischemic injury. The optimal treatment time after ischemic injury and dose were determined, respectively, as follows： （1） 2.0 hours and 10 mg/kg according to the results of toluidine blue staining; （2） 1.5 hours and 10 mg/kg according to early apoptotic ratio by flow cytometry; （3） 2.0 hours and 10 mg/kg according to immunohistochemical and western blot analysis; and （4） 1.5 hours and 10 mg/kg according to reverse transcription polymerase chain reaction. The present findings suggest that an intraperitoneal injection of 10 mg/kg picroside II 1.5-2.0 hours after cerebral ischemic injury in rats is the optimal dose and time for therapeutic benefit.

To Optimize the Therapeutic Dose and Time Window of Picroside II in Cerebral Ischemic Injury in Rats by Orthogonal Test

The paper aims to optimize the therapeutic dose and time window of picroside II by orthogonal test in cerebral ischemic injury in rats. The forebrain ischemia models were established by bilateral common carotid artery occlusion (BCCAO) methods. The successful models were randomly divided into sixteen groups according to orthogonal experimental design and treated by injecting picroside II intraperitonenally at different ischemic time with different dose. The concentrations of neuron-specific enolase (NSE), neuroglial marker protein S100B and myelin basic protein (MBP) in serum were determined by enzyme linked immunosorbent assay to evaluate the therapeutic effect of picroside II in cerebral ischemic injury. The results indicated that best therapeutic time window and dose of picroside II in cerebral ischemic injury were ischemia 1.5 h with 20 mg/kg body weight according to the concentrations of NSE, S100B and MBP in serum. It is concluded that according to the principle of lowest therapeutic dose with longest time window, the optimized therapeutic dose and time window are injecting picroside II intraperitonenally with 20 mg/kg body weight at ischemia 1.5 h in cerebral ischemic injury in rats.

The anti-inflammatory effect of picroside II and the optimizing of therapeutic dose and time window in cerebral ischemic injury in rats

The aim is to optimize the anti-inflammatory effect and the therapeutic dose and time window of picrosede II by orthogonal test in cerebral ischemic injury in rats. The forebrain ischemia models were established by bilateral common carotid artery occlusion (BCCAO) methods in 30 Wistar rats. The successful models were randomly divided into sixteen groups according to orthogonal experimental design and treated by injecting picroside II intraperitoneally at different ischemic time with different dose. The concentrations of aquaporins 4 (AQP4), matrix metalloproteinases9 (MMP9) and cyclooxygenase 2 (COX2) in serum and brain tissue were determined by enzyme linked immunosorbent assay to evaluate the therapeutic effect of picroside II in cerebral ischemic injury. The best therapeutic time window and dose of picroside II in cerebral ischemic injury were 1) ischemia 2.0 h with 20 mg/kg and 1.5 h with 20 mg/kg body weight according to the concentration of AQP4 in serum and brain tissue;2) ischemia 1.5 h with 20 mg/kg and ischemia 2.0 h with 20 mg/kg according to the concentrations of MMP9 in serum and brain tissue;and 3) ischemia 1.5 h with 10 mg/kg and ischemia 1.5 h with 20 mg/kg according to the concentrations of COX2 in serum and brain tissue respectively. According to the principle of the lowest therapeutic dose with the longest time window, the optimized therapeutic dose and time window were injecting picroside II intraperitoneally with 10 - 20 mg/kg body weight at ischemia 1.5 - 2.0 h in cerebral ischemic injury.

The Neuroprotective Effect of Picroside II and Its Best Therapeutic Dose and Time Window in Cerebral Ischemic Injury in Rats

Objective: To study the neuroprotective effect of picrosede II and explore the best therapeutic dose and time window according to orthogonal design in cerebral ischemic injury in rats. Methods: The forebrain ischemia rat models were established by bilateral common carotid artery occlusion (BCCAO) method. The successful models were randomly grouped according to orthogonal experimental design and treated by injecting picroside II intraperitoneally at different ischemic time with different doses. The contents of neuron-specific enolase (NSE), neuroglial marker protein S100B and myelin basic protein (MBP) in serum and brain tissue were determined by enzyme linked immunosorbent assay (ELISA) to evaluate the therapeutic effect of picroside II in cerebral ischemic injury. Results: The best therapeutic time window and dose of picroside II in cerebral ischemic injury may be 1) ischemia 1.5 h with 20 mg/kg and ischemia 1.5 h with 10 mg/kg body weight according to the content of NSE in serum and brain tissue respectively, 2) ischemia 1.5 h with 20 mg/kg according to the content of S100B in both serum and brain tissue, and 3) ischemia 1.5 h with 20 mg/kg and ischemia 1.5 h with 10 mg/kg according to the content of MBP in serum and brain tissue respectively. Conclusion: Based on the principle of the minimization of therapeutic drug 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 10-20 mg/kg body weight at ischemia 1.5 h in cerebral ischemic injury in rats.

The effect of picroside II on the expressions of AQP4, MMP9 and COX2 in cerebral ischemic injury in rats

The aim is to study the neuroprotective effect and optimize the therapeutic dose and time window of picrosede II by orthogonal test in cerebral ischemic injury in rats. The forebrain ischemia models of rats were established by bilateral common carotid artery occlusion (BCCAO) methods. The successful models were randomly grouped according to orthogonal experimental design and treated by injecting picroside II intraperitonenally with different therapeutic dose at different ischemic time. The contents of aquaporins 4 (AQP4), matrix metalloproteinases 9 (MMP 9) and cyclooxygenase 2 (COX2) in brain tissue were determined by enzyme linked immunosorbent assay to evaluate the therapeutic effect of picroside II on cerebral ischemic injury. The results indicated that the best therapeutic time window and dose of picroside II in cerebral ischemic injury should be 1) ischemia 1.5 h with 20 mg/kg body weight according to the content of AQP4;2) ischemia 2.0 h with 20 mg/kg according to the content of MMP9;and 3) ischemia 1.5 h with 20 mg/kg according to the content of COX2 inbrain tissue. It is concluded that the optimized therapeutic dose and time window is injecting picroside II peritonenally with 10 - 20 mg/kg body weight at ischemia 1.5 - 2.0 h in cerebral ischemic injury according to the principle of lowest therapeutic dose with longest time window.

Picroside Ⅱ promotes HSC apoptosis and inhibits the cholestatic liver fibrosis in Mdr2^(−/−)mice by polarizing M1 macrophages and balancing immune responses

Liver fibrosis is characterized by chronic inflammatory responses and progressive fibrous scar formation.Macrophages play a central role in the pathogenesis of hepatic fibrosis by reconstructing the immune microenvironment.Picroside Ⅱ(PIC Ⅱ),extracted from Picrorhizae Rhizoma,has demonstrated therapeutic potential for various liver damage.However,the mechanisms by which macrophage polarization initiates immune cascades and contributes to the development of liver fibrosis,and whether this process can be influenced by PIC Ⅱ,remain unclear.In the current study,RNA sequencing and multiple molecular approaches were utilized to explore the underlying mechanisms of PIC Ⅱ against liver fibrosis in multidrug-resistance protein 2 knockout(Mdr2^(−/−))mice.Our findings indicate that PIC Ⅱ activates M1-polarized macrophages to recruit natural killer cells(NK cells),potentially via the CXCL16-CXCR6 axis.Additionally,PIC Ⅱ promotes the apoptosis of activated hepatic stellate cells(aHSCs)and enhances the cytotoxic effects of NK cells,while also reducing the formation of neutrophil extracellular traps(NETs).Notably,the anti-hepatic fibrosis effects associated with PIC Ⅱ were largely reversed by macrophage depletion in Mdr2^(−/−)mice.Collectively,our research suggests that PIC Ⅱ is a potential candidate for halting the progression of liver fibrosis.

Optimization of microwave-assisted extraction for picroside I and picroside II from Picrorrhiza kurroa using Box-Behnken experimental design

The response surface methodology was employed to study the optimization of microwave-assisted extraction of picroside I and picroside II from Picrorrhiza kurroa Royle rhizomes. The effects of solid to solvent ratio, and extraction temperature, time and solvent on the yields ofpicroside I and picroside II have been investigated using Box-Behnken experimental design. The experimen- tal data were fitted to second-order polynomial equations using multiple regression analysis and analyzed using the appropriate statistical method. By solving the regression equation and analyzing 3-D plots, the optimum extraction conditions were found to be： solid to solvent ratio, 10 ： 90 （w/v）; temperature, 60 ~C; and extraction time, 60 s. Under the optimal conditions, the yields of picroside I and picroside II are 41.23 and 6.12 mg.gI feed respectively, which are in good agreement with the predicted values. The ratio of solid to solvent significantly affects the yields of picroside I and picroside II. Application of microwave- assisted extraction of picroside I and picroside II from P kurroa would dramatically reduce extraction time and solvent consumption.