[Astragaloside IV attenuates cerebral ischemia and reperfusion injury and reduces activation of NLRP3 inflammasome and NF-κB phosphorylation in rats following a transient middle cerebral artery occlusion].

The present study was aimed to investigate the protective effect and anti-inflammation mechanism of astragaloside IV (AST-IV) on cerebral ischemia and reperfusion injury. Following the establishment of cerebral ischemia and reperfusion model in rats by modified suture method, neurological deficit scores and cerebral infarct volume were used to evaluate the pharmacological effect of AST-IV against cerebral ischemia-reperfusion injury. Western blot was used to detect the expression levels of NLRP3, pro-Caspase-1, Caspase-1, pro-IL-1ß, IL-1ß, pro-IL-18, IL-18, phosphorylated and total nuclear factor kappa B (NF-κB)/p65 protein in the brain tissue. The results showed that compared with model group, the intervention of AST-IV decreased the neurological deficit scores, reduced the cerebral infarct volume, decreased the levels of NLRP3, Caspase-1, pro-IL-1ß, IL-1ß, pro-IL-18 and IL-18, and inhibited the expression of phosphorylated NF-κB in brain tissue. The results suggest that AST-IV has a protective effect against cerebral ischemia and reperfusion injury, and its mechanism is related to inhibiting the phosphorylation of NF-κB and NLRP3 inflammasome activation.

[Effect of astragaloside â £ combined with Panax notoginseng saponins on cerebral ischemia-reperfusion injury and study of pharmacokinetics in rats].

The aim is to study the effect of astragaloside Ⅳ (AST Ⅳ) combined with Panax notoginseng saponins (PNS) on cerebral ischemia-reperfusion injury, and to probe the synergistic mechanism through the pharmacokinetics of the four major components such as AST Ⅳ, ginsenoside Rg1 (Rg1), ginsenoside Rb1 (Rb1), notoginsenoside R1 (R1) in cerebral ischemia-reperfusion rats. Following the establishment of cerebral ischemia/reperfusion model in rats by modified suture method, neurological function score, cerebral infarction area and pathomorphology were used to evaluate the pharmacological effect that the combination of AST Ⅳ and PNS antagonized cerebral ischemia-reperfusion injury; the contents of AST Ⅳ, Rg1, Rb1, R1 in rat plasma of different time points were determined with ultra performance liquid chromatography tandem massspectrometry (UPLC-MS/MS), pharmacokinetic parameters were calculated and pharmacokinetics changes of the main effective components were analyzed. The results showed that AST Ⅳ, PNS alone and their combination could reduce the cerebral infarction area of rats, relieve the behavioral scores of neurologic deficit, improve the pathological changes after cerebral ischemia, the effects of the combination were better. Among AST Ⅳ, Rg1, Rb1, R1, the area under the curve (AUC) was significantly increased, the mean residence time of (MRT0-t) was delayed, the peak concentration (Cmax) was significantly raised, the apparent volume of distribution (Vz/F) was reduced, and the clearance rate in vivo was significantly slowed. It suggested that AST Ⅳ combined with PNS has synergistic enhancement on anti-cerebral ischemia/reperfusion injury, moreover, make the pharmacokinetic behavior of the main effective components change, the mechanism may be associated with prolonging the retention time of the effective components in cerebral ischemia condition, elevating the bioavailability.

[In vitro microdialysis recoveries of nine active ingredients in Mahuang decoction].

To detect the in vitro probe microdialysis recoveries based on an HPLC-DAD method for simultaneous quantification of nine active ingredients (ephedrine, pseudoephedrine, methylephedrine, amygdalin, liquiritin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde and glycyrrhizic acid) in Mahuang decoction, which provides reference for in vivo pharmacokinetic study. The concentrations of nine active ingredients in dialysate were detected by HPLC-DAD, to investigate the effect of flow rates (incremental method and subtraction method) and intraday stability of the probe recoveries and medium concentrations on the recoveries. Nine active ingredients could be well separated in 52 min. At the perfusion rate of 1.0 µL x min(-1), the relative recoveries of ephedrine, pseudoephedrine, methylephedrine, amygdalin, liquiritin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde and glycyrrhizic acid were (50.95 ± 0.82)%, (52.74 ± 1.13)%, (51.29 ± 0.51)%, (32.56 ± 0.84)%, (45.36 ± 0.83)%, (70.94 ± 0.99)%, (69.98 ± 2.30)%, (71.68 ± 0.63)%, and (22.14 ± 0.48)%, respectively. And the probe kept steady in 7 hours. At the same medium concentration, the probe recoveries decreased exponentially with the increase in flow rates. The recoveries of seven ingredients detected by these two methods were similar at certain flow rates, except for amygdalin and cinnamaldehyde. At the same flow rate, the relative recoveries of cinnamyl alcohol, cinnamic acid and cinnamaldehyde changed greatly (9.55%-16.2%) and the others six ingredients had less change (3.27%-5.71%) with the changes in medium concentrations. Microdialysis method could be used to detect the in vitro recoveries of nine ingredients in Mahuang decoction. Reverse dialysis method could be used for the in vivo probe recovery calibration of ephedrine, pseudoephedrine, methylephedrine, liquiritin, cinnamyl alcohol and cinnamic acid at the flow rate of 2.0 µL x min(-1).

[Effects of Buyang Huanwu Decoction and its alkaloids and glycosides on aortic intimal hyperplasia and expression of proliferating cell nuclear antigen in rats with aortic intimal injuries].

OBJECTIVE: To explore the effects of alkaloids and glycosides extracted from Buyang Huanwu Decoction (BYHWD), a compound of traditional Chinese herbal medicine, on aortic intimal hyperplasia and the expression of the proliferating cell nuclear antigen (PCNA) in rats with aortic intimal injuries. METHODS: The vessel restenosis model was established by denuding aortic endothelium with domestic balloon catheter in rats. Drugs were administered intragastrically on the first day after operation. The injured segments of aorta were taken on the fifteenth day after operation to determine the degree of intimal hyperplasia and observe the expression of PCNA. RESULTS: Aortic intimal hyperplasias were very obvious on the fifteenth day after operation. The media hyperplasias in the drug-treated groups were not significant (P>0.05), but the intimal hyperplasia were remarkable as compared with that in the sham-operated group (P<0.01). The degrees of intimal hyperplasia in BYHWD, alkaloids, glycosides and atorvastatin groups were less than that in the untreated group (P<0.01). There was significant difference of PCNA expression between the untreated group and the sham-operated group (P<0.01). The expressions of PCNA in alkaloids, glycosides, and atorvastatin groups were higher than that in the sham-operated group (P<0.01, P<0.05), but still lower than that in the untreated group (P<0.01). The expression of PCNA in BYHWD group was higher than that in the sham-operated group (P<0.01), and no significant difference was found between the BYHWD group and the untreated group (P>0.05). CONCLUSION: Alkaloids and glycosides extracted from BYHWD can inhibit intimal hyperplasia induced by denuding arterial endothelium with domestic balloon catheter in rats. Alkaloids and glycoside may be among basal substances in BYHWD inhibiting intimal hyperplasia of blood vessel, the effect of which may relate to down-regulating the expression of PCNA.

[Effects of Panax notoginseng saponins on mRNA expressions of interleukin-1 beta, its correlative factors and cysteinyl-aspartate specific protease after cerebral ischemia-reperfusion in rats].

OBJECTIVE: To investigate the effects of Panax notoginseng saponins (PNS) on mRNA expressions of interleukin-1 beta (IL-1 beta), interleukin-1 receptor type I (IL-1RI), interleukin-1 receptor antagonist (IL-1ra), intercellular adhesion molecule-1 (ICAM-1), cysteinyl-aspartate specific protease-1 (caspase-1), caspase-3 and caspase-8 after cerebral ischemia-reperfusion in rats. METHODS: Focal cerebral ischemia reperfusion in rats was induced by the method of nylon monofilament via the internal carotid artery. PNS was administered intraperitoneally respectively five minutes before cerebral ischemia and twelve hours after cerebral ischemia. After cerebral ischemia for two hours followed by reperfusion for twenty two hours, the mRNA expressions of IL-1 beta, IL-1RI, IL-1ra, ICAM-1, caspase-1, caspase-3 and caspase-8 in brain tissue were determined by reverse transcription polymerase chain reaction assay. RESULTS: After cerebral ischemia for two hours followed by reperfusion for twenty two hours, the mRNA expression levels of IL-1 beta, IL-1RI, IL-1ra, ICAM-1, caspase-1, caspase-3 and caspase-8 in brain tissue in the untreated group were obviously elevated as compared to those in the sham-operation group (P<0.05 or P<0.01). The mRNA expression levels of IL-1 beta, IL-1RI, IL-1ra in brain tissue in the PNS group were lower than those in the untreated group, but higher than those in the sham-operation group, and without statistical differences as compared to those in the sham-operation group and in the untreated group (P>0.05). The mRNA expression level of caspase-3 in brain tissue in the PNS group was significantly lower than that in the untreated group (P<0.05), but PNS had no effect on the mRNA expression levels of ICAM-1, caspase-1 and caspase-8 in brain tissue. CONCLUSION: PNS can inhibit the mRNA expression of caspase-3, slightly inhibit the mRNA expressions of IL-1 beta and its correlative inflammatory factors in brain tissue. The protective effects of PNS on cerebral injury induced by ischemia-reperfusion may be related to inhibiting the mRNA expressions of caspase-3, IL-1 beta and its correlative inflammatory factors in brain tissue.

Synergism and mechanism of Astragaloside IV combined with Ginsenoside Rg1 against autophagic injury of PC12 cells induced by oxygen glucose deprivation/reoxygenation.

The aim of this study was to explore the effect by which the combination of Astragaloside IV (AST IV) and Ginsenoside Rg1 (Rg1) resisted autophagic injury in PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). We studied the nature of the interaction between AST IV and Rg1 that inhibited autophagy through the Isobologram method, and investigated the synergistic mechanism via the PI3K I/Akt/mTOR and PI3K III/Becline-1/Bcl-2 signaling pathways. Our results showed that, based on the 50% inhibiting concentration (IC50), AST IV combined with Rg1 at a 1:1 ratio resulted in a synergistic effect, whereas the combination of the two had an antagonistic effect on autophagy at ratios of 1:2 and 2:1. Meanwhile, AST IV and Rg1 alone increased cell survival and decreased lactate dehydrogenase (LDH) leakage induced by OGD/R, reduced autophagosomes and the LC3 II positive patch, down-regulated the LC3 II/LC3 I ratio and up-regulated the p62 protein; the 1:1 combination enhanced these effects. Mechanistic study showed that Rg1 and the 1:1 combination increased the phosphorylation of PI3K I, Akt and mTOR; the effects of the combination were greater than those of the drugs alone. AST IV and the 1:1 combination suppressed the expression of PI3K III and Becline-1, and the combination elevated Bcl-2 protein expression; the effects of the combination were better than those of the drugs alone. These results suggest that after 2 h-OGD followed by reoxygenation for 24h, PC12 cells suffer excessive autophagy and damage, which are blocked by AST IV or Rg1; moreover, the combination of AST IV and Rg1 at a 1:1 ratio of their IC50 concentrations has a synergistic inhibition on autophagic injury. The synergistic mechanism may be associated with the PI3K I/Akt/mTOR and PI3K III/Becline-1/Bcl-2 signaling pathways.

[Effect of active fraction of buyang huanwu decoction on caspase expression in rats after focal cerebral ischemic reperfusion].

OBJECTIVE: To investigate effect of the four kinds of active fractions extracted from Buyang Huanwu Decoction (BYHWD) on caspase expression in rats after focal cerebral ischemic reperfusion. METHODS: The focal cerebral ischemia/reperfusion model rats were established by middle cerebral artery occlusion for 2 hrs followed with reperfusion for 46 hrs. Before and at 12th, 24th and 36th hour after cerebral ischemia, the rats were administered with alkaloid, glycoside, polysaccharide and aglycone from BYHWD respectively, and nimodipine was given as control medicine to observe the effect of them on protein expression of caspase-1, caspase-3 and caspase-8 using immunohistochemical method. RESULTS: Protein expression of caspase-1 and caspase-3 increased in the injured lateral brain tissue after cerebral ischemia/reperfusion. Caspase-1 expression were observed mainly in choroids, while caspase-3 expression in hippocampus, cortex and medulla. All the four kinds of active fractions from BYHWD could inhibit caspase-1 expression, while nimodipine couldn't. Caspase-3 expression in hippocampus and medulla could be inhibited by alkaloid, that in hippocampus, cortex and medulla could be inhibited by glycoside and aglycone, and that in hippocampus and medulla by nimodipine, however, polysaccharide showd no effect on it. As for caspase-8 expression, no effect on it was shown in all groups. CONCLUSION: Alkaloid, glycoside, polysaccharide and aglycone from BYHWD could relieve the inflammatory reaction occurred after cerebral ischemia/reperfusion by inhibiting caspase-1 expression to decrease production of inflammatory cytokine. Alkaloid, glycoside and aglycone could reduce neuronal apoptosis by inhibiting caspase-3 expression to antagonize the delayed neuronal death after cerebral ischemia. The 4 kinds of active fractions may be the main material basis for BYHWD in preventing ischemia/reperfusion cerebral injury.

Effects of the main active components combinations of Astragalus and Panax notoginseng on energy metabolism in brain tissues after cerebral ischemia-reperfusion in mice.

BACKGROUND: Astragalus and Panax notoginseng are traditional Chinese medicines used for the treatments of cardio-cerebrovascular ischemic diseases, astragaloside IV (AST IV) and ginsenoside Rg1 (Rg1), ginsenoside Rb1 (Rb1), notoginsenoside R1 (R1) are their active components. OBJECTIVE: The purpose of this work was to investigate the effect of AST IV combined with Rg1, Rb1, R1 on energy metabolism in brain tissues after cerebral ischemia-reperfusion in mice. MATERIALS AND METHODS: C57BL/6 mice were randomly divided into 11 groups, treated for 3 days. At 1 h after the last administration, the model of cerebral ischemia-reperfusion injury was established, and brain tissues were detected. RESULTS: All drugs increased the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and the level of total adenine nucleotides (TAN), the combinations increased energy charge (EC), the effects of four active components combination were better. The phosphorylation of AMP-activated protein kinaseα1/2 (p-AMPKα1/2) was increased in AST IV, R1, four active components combination, AST IV + Rg1 and AST IV + R1 groups, the increased effect of four active components combination was greater than that of the active components alone and AST IV + Rb1. All drugs increased glucose transporter 3 (GLUT3) mRNA and protein, and the increases of four active components combination were more obvious than those of the active components alone or some two active components combinations. CONCLUSION: Four active components combination of Astragalus and P. notoginseng have the potentiation on improving of energy metabolism, the mechanism underlying might be associated with promoting the activation of AMPKα1/2, enhancing the expression of GLUT3, thus mediating glucose into nerve cells, increasing the supply and intake of glucose.

Effects of the Combination of the Main Active Components of Astragalus and Panax notoginseng on Inflammation and Apoptosis of Nerve Cell after Cerebral Ischemia-Reperfusion.

Astragalus and Panax notoginseng are commonly used to treat cardio-cerebrovascular diseases in China and are often combined together to promote curative effect. We speculate that the enhancement of the combination on anticerebral ischemia injury may come from the main active components. The purpose of this work was to probe the effects and mechanisms of Astragaloside IV (the active component of Astragalus) combined with Ginsenoside Rg1, Ginsenoside Rb1, and Notoginsenoside R1 (the active components of P. notoginseng) to antagonize ischemia/reperfusion (I/R) injury via inflammation and apoptosis. C57BL/6 mice were randomly divided into sham, model, Astragaloside IV, Ginsenoside Rg1, Ginsenoside Rb1, Notoginsenoside R1, four active components combination, and Edaravone groups. After administration for 3 days, bilateral common carotid arteries (CCA) were occluded with artery clip for 20[Formula: see text]min followed by reperfusion for 24[Formula: see text]h. Our results showed that the survival rate of nerve cell in hippocampal CA1 decreased while the apoptotic rate increased, and the level of caspase-3 protein in brain tissues was elevated, the expressions of TNF-a, IL-1, and ICAM-1 mRNA as well as phosphorylated nuclear factor kappa B (NF-κB) inhibitor protein α (p-IκBa) in brain tissues were up-regulated, and the nuclear translocation rate of NF-κB was raised. Additionally, the protein expressions of phosphorylated tyrosine kinase 1 (p-JAK1), phosphorylated signal transducer and activator of transcription-1 (p-STAT1), glucose regulated protein 78 (GRP78), caspase-12, and phosphorylated c-Jun N-terminal kinases 1/2 (p-JNK1/2) in brain tissues were also significantly strengthened after I/R for 24 h. All drugs could increase neurocyte survival rate in hippocampal CA1, decrease the apoptotic rate, and inhibit caspase-3 protein expression, in contrast, the effects of four active components combination were better than those of active components alone. In addition, Astragaloside IV and Ginsenoside Rg1 could down-regulate the level of TNF-α, and ICAM-1 mRNA, respectively, Notoginsenoside R1 reduced both TNF-α and ICAM-1 mRNA, and the combination of the 4 effective components had inhibitory effects on the expressions of TNF-α, IL-1ß, and ICAM-1 mRNA. Astragaloside IV, Ginsenoside Rg1, Notoginsenoside R1, and 4 effective components combination were able to restrain the phosphorylation of IκBα, and relieve the nuclear translocation rate of NF-κB. Moreover, the effects of the combination are greater than those of active components alone. All drugs could suppress the phosphorylation of JAK1 induced by I/R; meanwhile the expression of p-STAT1 exhibited a decrease in Ginsenoside Rg1 and four active components combination groups. The decreases of p-JAK1 and p-STAT1 in the four active components combination group were more obvious than those in active components alone groups. Astragaloside IV, Ginsenoside Rg1, and Notoginsenoside R1 further augmented GRP78 expression caused by I/R, Notoginsenoside R1 attenuated caspase-12 protein expression, Astragaloside IV and Ginsenoside Rg1 lessened the phosphorylation of JNK1/2, and the four active components combination was capable of up-regulating GRP78 protein while down-regulating the expressions of caspase-12 and p-JNK1/2. Similarly, the effects of the four active components combination were greater than those of effective components alone. These suggested that the combination of the main active components of Astragalus and Panax notoginseng could strengthen protective effects on cerebral ischemia injury via anti-apoptosis and anti-inflammation, and the mechanisms might be associated with restraining the activation of NF-κB and JAK1/STAT1 signal pathways and regulating endoplasmic reticulum stress (ERS) after cerebral ischemia.

Autophagy in cerebral ischemia and the effects of traditional Chinese medicine.

Autophagy is a lysosome-mediated degradation process for non-essential or damaged cellular constituents, playing an important homeostatic role in cell survival, differentiation and development to maintain homeostasis. Autophagy is involved in tumors as well as neurodegenerative, cardiovascular and cerebrovascular diseases. Recently, active compounds from traditional Chinese medicine (TCM) have been found to modulate the levels of autophagy in tumor cells, nerve cells, myocardial cells and endothelial cells. Ischemic stroke is a major cause of neurological disability and places a heavy burden on family and society. Regaining function can significantly reduce dependence and improve the quality of life of stroke survivors. In healthy cells, autophagy plays a key role in adapting to nutritional deprivation and eliminating aggregated proteins, however inappropriate activation of autophagy may lead to cell death in cerebral ischemia. This paper reviews the process and the molecular basis of autophagy, as well as its roles in cerebral ischemia and the roles of TCM in modulating its activity.

Effects of Astragaloside IV combined with the active components of Panax notoginseng on oxidative stress injury and nuclear factor-erythroid 2-related factor 2/heme oxygenase-1 signaling pathway after cerebral ischemia-reperfusion in mice.

BACKGROUND: Astragalus and Panax notoginseng are traditional Chinese Medicines used for the treatments of ischemic cerebrovascular disease, being often combined together in China and achieving a good effect. OBJECTIVE: The objective of this study is to investigate the effects of astragaloside-IV (AST-IV) (the effective component of Astragalus) combined with ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1 (the effective components of P. notoginseng) on oxidative stress injury after cerebral ischemia-reperfusion in mice, and to explore the mechanisms through nuclear factor-erythroid 2-related factor-2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway. MATERIALS AND METHODS: C57BL/6 mice were randomly grouped after treated for 3 days, the model of cerebral ischemia-reperfusion injury was established, and the brain tissues were detected. RESULTS: AST-IV combined with ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1 could increase significantly the survival rate of nerve cell; decrease the contents of malondialdehyde, nitric oxide, increase the activity of superoxide dismutase and the level of glutathione; Nrf2 was down-regulated in the cytoplasm while up-regulated in nucleus, nuclear translocation rate raised as well as HO-1 messenger ribonucleic acid and protein expressions increased. The effects of four active components combination were better than those of the active components alone. CONCLUSION: Active components of Astragalus and P. notoginseng had the effects against cerebral ischemia-reperfusion injury, which were related to the antioxidative stress after cerebral ischemia-reperfusion. AST-IV combined with ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1 could strengthen the antagonism effects on ischemia-reperfusion and oxidative stress injury, the mechanism underlying might be associated with jointly activating Nrf2/HO-1 signaling pathway after cerebral ischemia-reperfusion.

Effect of total saponins of "panax notoginseng root" on aortic intimal hyperplasia and the expressions of cell cycle protein and extracellular matrix in rats.

AIM OF THE STUDY: the effect of total saponins of "panax notoginseng root" on aortic intimal hyperplasia and the expressions of cell cycle protein and extracellular matrix in rats MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into sham-operated, control, TSPN and atorvastatin group. Rat aorta intima in all groups were injured by insertion of domestic balloon catheter into the aortae except sham-operated rats. Drugs were administrated orally from the second day after vascular injury and continued for 14 days. The injured segments of aortae were collected on the sixteenth day after operation to observe the morphological changes of vascular structure and to examine the expressions of proliferating cell nuclear antigen(PCNA), cyclinD1, cyclinE, collagen I(Col-I), fibronect(FN), matrix metalloproteinase-9(MMP-9) and tissue inhibitor metalloproteinase-1(TIMP-1). RESULTS: TPNS significantly inhibited the vascular intimal hyperplasia. TPNS significantly lowered the expression of PCNA, cyclinE, cyclinD1, FN and MMP-9. TPNS had no significant impacts on the expression of Col-I and TIMP-1. CONCLUSIONS: Our studies indicated that TSPN could inhibit vessel restenosis after vascular intimal injury, and its mechanisms may be related to the blockage of the excessive proliferation of VSMC, the reduction of ECM protein deposition in the endometrium, and the degradation of ECM protein.

Inhibition of aortic intimal hyperplasia and cell cycle protein and extracellular matrix protein expressions by BuYang HuanWu Decoction.

AIM OF THE STUDY: The inhibitive effect of BuYang HuanWu Decoction (BYHWD) and its major components on vascular intimal hyperplasia and the expressions of cell cycle protein and extracellular matrix protein. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into sham-operated, control, alkaloid, glycoside, BYHWD and atorvastatin groups. Rat aorta intima in all groups were injured by insesion of domestic balloon catheter into the aortae except sham-operated rats. Drugs were administrated orally from the second day after vascular injury and continued for 14 days. The injured segments of aortae were collected on the sixteenth day after operation to observe the morphological changes of vascular structure and to examine the expressions of proteins in vascular cells associated with cell cycle including proliferating cell nuclear antigen(PCNA), cyclinD(1) and cyclinE, and extracellular matrix(ECM) proteins including collagen I (Col-I) and fibronectin (FN), further to discover the involved biologically active substances and the potential mechanisms. RESULTS: Alkaloid and glycosid isolated from BYHWD were more effective than BYHWD in the inhibition of intimal hyperplasia and the expressions of PCNA, cyclinD(1), cyclinE, Col-I and FN, suggesting that alkaloid and glycoside may be the main components of BYHWD responsible for the observed inhibition of excessive hyperplasia of vascular intima. CONCLUSIONS: The mechanism associated with the anti-hyperplasia activity of BYHWD and its effective components may be related to the blockage of cell cycles of VSMC, and the inhibition of the ECM protein synthesis, even the increased degradation of ECM proteins.