| BackgroundCerebrovascular disease which is one of the three major diseases causes human death, the morbidity rate of cerebrovascular disease in our country is higher than the average level in other countries. Therefore, the study of ischemic cerebrovascular disease is one of the important research topics.Total Saponins of Panax Notoginseseng have the effect on promotion blood circulation to remove blood stasis, which is widely used in the treatment of ischemic cerebrovascular disease. Pharmacological studies showed that PNS is the main effective component of Chinese medicine notoginseng, PNS have obvious effect on repairment and regeneration of the vascular endothelium. Gene expression profile chip shows Total Saponins of Panax Notoginseseng couldincrease endothelial cells under hypoxia state of low sugar the expression of TGF-beta, SMAD5, ID 1.The results suggested TGF-beta-SMAD5/ID1 signaling pathway could be one of the main targets of protecting endothelial cells, but the specific mechanism is still unknown. Therefore, this study from TGF-beta-SMAD5/ID1 signaling pathway used brain microvascular endothelial cells in vitro, using oxygen-glucose deprivation model,and observed the molecular mechanism of protectionendothelial cells, anti-apoptoticand and provide experimental basis for its clinical application.ObjectiveIn this experiment,cerebral microvascular endothelial cellsare taken as the starting point. A model of ischemic damage due to oxygen and glucose deprivation was prepa red. From TGF-beta-SMAD5/ID1 apoptosis signaling pathways/we observed the function and effecton PNS protectiveendothelial cells andanti-apoptosis.The aim of this study is to explain the molecularmechanism and the therapeutic targets.Methods1. Using ubiquitous methods to obtain rat brain microvascular endothelial cells, culturing, purifying and reproducing them.Using the method of oxygen-glucose deprivation to emulate cell ischemic injury.2. The cerebral microvascular endothelial cells were cultured to 3rd generation. The cells were randomly divided into 4 groups:normal group, model group, notoginseng total saponin group, notoginseng total saponin+blockers. In addition to the normal group, OGD were used in other groups. The PNS group was used 22 mu g/ml total saponins of panax notoginseng in the process of, the PNS+blockers group was used 22 mu g/ml total saponins of panax notoginseng and 1.8 nM blocker K02288 (a type I highly selective BMP receptor inhibitor).3. Observing the protective effect and mechanism of PNS onendothelial cells with ischemic injury.The endothelial cell apoptosis were detectedby flow cytometry.4. The expression of TGF-beta was detected by ELISA,the expression of ALK1 mRNA, ALK1 protein, SMAD5 mRNA,SMAD5 protein and ID1 mRNA were detected by Western blot andReal-Time PCR.Observing of regulation of PNS on ischemia endothelial cells from TGF-beta SMAD5/ID1 signaling pathways5. The expression of TSP-1 and VEGF-B were detected by ELISA.Explain the anti-apoptosis mechanism of PNS onendothelial cells with ischemic injuryResult1. The effect of PNS on cell apoptosisCompared with the normal group, the rate of cell apoptosis in model group significantly increased (P< 0.01); Compared with model group, the rate of cell apoptosis in PNS group significantly decreased (P< 0.01).Compared with PNS+blockers, the rate of cell apoptosis in PNS group decreased little, there was no statistically significant difference (P> 0.05).2.The effect of PNS on the expression of TGF-beta/Smads/iDl signaling pathwaysCompared with the normal group, the expression of TSP-1 in model group increased significantly(P< 0.05); Compared with model group, the expression of TSP-1 in PNS group decreased significantly (P> 0.05).Compared with the normal group, the expression of ALK1 mRNA in model group significantly increased (P< 0.01); compared with model group, the expression of ALK1 mRNA increased little in PNS group(P>0.05).ALK1 protein expression were detectedby Western blotting, ALK1 protein expression in normal groupwas less, after ischemia injury, the expression of ALK1 protein increased in model group (P< 0.05).Compared with model group, the expression of ALK1 protein increased in PNS group (P< 0.05).Compared with the normal group, the expression Smad5 mRNA in model groupsignificantly increased (P< 0.01); Compared with model group, the expression Smad5 mRNA inPNS groupsignificantly increased (P< 0.01).After ischemic injury, the expression of Smad5 protein in model group significantly increased (P< 0.05); Compared with model group, the expression of Smad5 protein increased little in PNS group(P> 0.05).Compared with the normal group, the expression of idl mRNA in model group was significantly decreased (P< 0.01); Compared with model group, the expression of idl mRNA in PNS group was significantly increased (P< 0.01).3.The effect of PNS on the expression of TSP-1 and VEGFCompared with the normal group, the expression of TSP-1 in model group increased significantly(P< 0.01); Compared with model group, the expression of TSP-1 in PNS group decreased significantly (P< 0.01).Compared with PNS+blockers, the expression of TSP-1 in PNS group decreased significantly (P< 0.01).Compared with the normal group, the expression of VEGF in model group increased significantly(P< 0.01); Compared with model group, the expression of VEGFin PNS group decreased significantly (P< 0.05).There is no different between PNS group and PNS+blockers in the expression of VEGF(P> 0.05).ConclusionLEndothelial cell apoptosis increased significantly after ischemia injury,PNS inhibited apoptosis of endothelial cells and reduced the damage of endothelial cells under oxygen deficient condition.2.The expression of ALK1 mRNA, ALK1 protein, Smad5 mRNA, Smad5 protein,idlmRNA were increased significantly after ischemia injury.PNS increased the expression of SmadSmRNA and protected cerebrovascular endothelial cells after ischemic injury3. PNS decreased the expression of TSP-1 and increased the expression of VEGF-B.BMP can partly block the effects.The results suggests PNS have an effect on anti-apoptosis of endothelial cells with ischemic injury andprotected cerebrovascular endothelial cells after ischemic injury. TGF-beta/Smad5/Idl signaling pathway may be one of the molecular mechanisms of PNS protective effect. |
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