| BackgroundCatalpol is one of the main active ingredients from Rehmannia Root.Previous studies have confirmed its neuroprotective effect in the acute phase of cerebral ischemia,can improve cerebral ischemia rats neurobehavioral function,promote angiogenesis and adult neural stem cells proliferation of the area around ischemia.However,the effects of delayed administration of catalpol on cerebral ischemia rats and differentiation of neural stem cells have not been studied.Because of the present situation of the treatment of cerebral ischemia disease drug shortage,and currently accepted effective venous thrombolysis drug t PA treatment time window stenosis(3-4.5 h),in the period of time most patients can not arrive at the hospital in time,catalpol is a potential protective agent in the treatment of ischemic brain injury.If it can protect ischemia and surrounding tissues outside the t PA treatment window,it is of great scientific value and clinical significance to study its pharmacodynamic effect and differentiation effect on newborn neural stem cells.ObjectiveTo observe the effects of delayed administration of catalpol on neurobehavioral effects,neurogenesis in rats after focal permanent cerebral ischemia.and its effect on the differentiation of neural stem cells.Methods1.The SD rats were randomly divided into 6 groups,were divided into sham group,model group,6 h for the first time administration with catalpol group(5 mg/kg and 10 mg/kg),4 d for the first time administration with catalpol group(5 mg/kg and 10 mg/kg).The model of focal permanent cerebral ischemia(p MCAO)was prepared by electrocoagulation.Catalpol was injected intraperitoneally in the treatment group and the same dose of normal saline in the model group,once a day for 7 days.Weight measurement,Bederson score and neurological deficit score(m NSS),muscle strength test were performed on each group at 1,4,7,14,21,28 days after administration.2.Brd U was injected intraperitoneally to label proliferation cells and animals were sampled after 7,14,21,28 days.The proliferation and survival of neural stem cells in SVZ region were detected by immunofluorescence double labeling of Brd U/Nestin.The differentiation of neural stem cells into astrocytes and neurons was detected by Brd U/GFAP,Brd U/DCX and Brd U/Neu N.Western Western Blot(WB)was used to detect the protein expression of Nestin,DCX in SVZ and GFAP,Neu N in the cortex of ischemic cerebral regions in each group.3.Due to the small number of SVZ in neonatal rats and it is difficult to separate,this study selected neonatal rat hippocampal neural stem cells within 24 hours of primary culture for purification and identification,Brd U labeled cell proliferation,and cck-8 was used to detect the effect of catalpol at different concentrations on neural stem cell viability.Adding differentiation medium to induce stem cell differentiation,immunofluorescence MAP-2,GFAP,MBP to detect cell differentiation,WB to detect the expression of differentiated phenotype-related proteins.Results1.We prepared the cerebral ischemia model successfully,and the infarct volume was 9.62±2.43%.Behavioral tests showed that the catalpol group increased the weight of cerebral ischemia rats in varying degrees,reduced the scores of Bederson and neurological impairment and improved the muscle strength test scores in cerebral ischemia rats,and there was a significant difference at 14 days(P <0.05,vs model),21 days basic neurological function recovery to control levels,there was no significant difference(P > 0.05,vs sham),but there was no significant difference between each dose group.2.Catalpol promotes the proliferation and survival of neural stem cells in the SVZ area of the injured side of focal permanent cerebral ischemia rats at different time points.Brd U/Nestin fluorescence results indicate the existence of adult neural stem cells 7 days and 14 days after cerebral ischemia,the number of astrocytes increased significantly(P<0.05,vs model),and the number of new neural stem cells and neurons increased significantly compared with the model group(P<0.05,vs model).The expression of DCX increased at 14 days.There was a significant difference between the 6 h,5 mg/kg dose group and the model group(P<0.05,vs model),and the other administration groups had significant differences compared with the sham operation(P<0.05,vs.sham).At 21 days,the expression of GFAP in each administration group decreased,and the expression of Nestin and Neu N increased.Compared with the model group,there were significant differences(P<0.05,vs model),which was basically consistent with the behavioral results.3.After 7 days,hippocampal neural stem cells could be proliferated into neurons with a diameter of about 200 μm,and the neurons presented double-labeling positive Brd U/Nestin.The concentration of catalpol that promotes the growth of neural stem cells selected by cck-8 is 0.1,1,10,100 Μm;different concentrations of catalpol were used to induce P3 generation neural stem cells to differentiate for 7 days.MAP-2 positive neurons,GFAP positive astrocytes and MBP positive oligodendrocytes were observed;10 μM Catalpol promoted NSCs to differentiate into neurons and 0.1 μM to oligodendrocytes.Conclusion1.Delayed administration of catalpol promotes body weight and improves neurobehavioral score in rats with focal permanent cerebral ischemia.2.Delayed administration of catalpol promoted the proliferation of neural stem cells in SVZ region on the injured side of focal permanent cerebral ischemia in rats,promoted the survival of neurons near the ischemic region,and reduced the proliferation of astrocytes.3.The primary cultured hippocampal neural stem cells have strong self-proliferation and multi-differentiation potential.Catalpol has a dose effect on the differentiation of neural stem cells into neurons and glial cells. |
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