Effect Of SSTF On Learnging And Memory Damage Induced By Aβ And Its Mechanism In Rat

Alzheimer's disease(AD) which is also called senile dementia is a key disease inducing dementia. AD has become a heavy burden to family and society with society entering aging. Many studies have been done to explore the pathogenesis, prevention and treatment of AD since Alois Alzheimer first reported AD. Though the pathogenesis has been unclear, the idea that Aβis the initiate factor has been accepted. Aβ25-35 is the functional peptide fragment of Aβ, experimnts have shown that AP25-35 can induce hippocampus neuron damage, decreased learning and memory both in vivo and in vitro. Now there are no high-efficacy agent for AD in clinic. Anti-cholinergic agents play the key role in clinic. More experiments focus on finding the new therapeutic target through exploring the pathogenesis. Flavonoids are secondary metabolite widespread distributing in nature. Flavonoids are important effective components of anti-oxidative plants. Scutellaria is a traditional Chinese herb. It has been shown that SSTF has anti-inflammatory, anti-immuno and myocardial protection against the damage induced by ischemia-reperfusion. Using bilateral injection Aβin hippocampus to make AD model, we observe the protection of SSTF, explore the mechanism of SSTF on oxidative stress, inflammatory, neuron apoptosis, supply the experimental material for anti-oxidative plants used for prevention and treatment to AD.Part one. Effect of SSTF on learning and memory damage induced by Aβin ratObjective:To explore the effect of SSTF on learning and memory damage induced by Aβin rat.Method:1 experiment group and management:100 male Wistar rats were randomly divided into six groups(control, model, SSTF-L, SSTF-M,SSTF-H, VE, n=16). The distilled water was administered (ig, once for 20d) to the control and the model group, SSTF (25mg/kg/d,50mg/kg/d, 100mg/kg/d ig, once for 20d) and VE(10mg/d) were administered in SSTF-L,SSTF-M,SSTF-H and VE groups respectively. On the eighth day saline or Aβwas injected to bilateral hippocampus in the control group or the model, SSTF and VE groups.One week after injection AβMorris water maze was performed for 6d. Other 4 rats were killed after injection Aβ3d, brain tissue were cut continually with 30μm thick to observe the needle path.2 model preparation:colloidal state Aβ(5μl,10μg) was injected in hippocampus CA1 region of rat(AP-3.5mm, ML±2mm, DV 2.7mm) using brain stereotaxis technique.3 Morris water maze test:One week after injection AβMorris water maze was performed for 6d(twice one day) to record the latency finding the hidden platform, on the 6th day, the platform was moved to record the times crossing the platform area, time staying in the platform quadrant, ratio of path in platform quadrant to the whole path during 2min.4 specimen management:3 rats of each group were infused with paraformaldehyde, brain tissue was performed external fixation, paraffin imbedding, HE, thionine Nissl body stain. Congo red stain was used to make sure the injection site.2 rats of each group were chosen to made specimen of electron microscope.5 Statistical analysis:SPSS11.5 software was used to make statistical analysis.Result:1 Morris test result:the latency of control rats in 3th,4th,5th,6th were 24.65,18.02,14.24,10.32s respectively, the latency of model in 4d were significantly longer than those of control. Compared with that of model, the latency of SSTF-M, SSTF-H and VE groups in 4d were significantly decreased.In model group, times crossing the platform(6.25), time staying in the platform quadrant(29.24s), ratio of path in platform quadrant to the whole path(32.30%) during 2min were significantly shorter than those in control(15.5,60.22s,62.31%). Compared with that of model those in SSTF-M, SSTF-H and VE groups were significantly increased.2 HE:There were 3-4 layers cell in hippocampus CA1 region in control, cell shape were clear, integrity with dentrite; while in model, there were an obvious neuron loss in CA1 region, cell quantity of CA1 region in model (56.13) was significantly decreased compared with that of control(142.63). Compared with that of model the cell quantity were significantly increased in SSTF-M(86.12), SSTF-H(126.25) and VE(87.10). The result of Nissl stain in hippocampuse was similiar with that of HE stain.Nissl body stain showed that cell quantity in model cortex(108) was significantly decreased compared with that of control(543.38), the cell quantity were obviously increased in SSTF-M(451.88), SSTF-H(534.75) and VE(463.88) groups compared with that of model.3 Ultra-structure showed that clear nuclear membrane, more cell organelle appeared in hippocampus CA1 region of control, while in model, cellular membrane was irregular, discontinuation, nuclear membrane was cloudiness. Obviously decreased cellular organelle, degenerated mitochonadria and apoptosis neuron also appeared in model group. The damage in SSTF-M, SSTF-H were significantly decreased. The severe damage appeared in cortex neuron of model.The damage in cortex were significantly decreased in SSTF-M, SSTF-H compared with that of model.Conclusion:It could be concluded that the injection of Aβin hippocampus CA1 region could cause severe damage in CA1 region and cortex neuron, decrease learning and memory ability. Administration of SSTF-M, SSTF-H and VE could improve neuron structure of hippocampus, cortex, improve learning and memory ability of rat. SSTF has a beneficial brain protection against the damage induced by Aβand there was a dose-dependent.Part two Anti-oxidative and anti-inflammatory effect of SSTF to learning and memory damage induced by Aβin ratObjective:To explore the relationship between anti-oxidative, anti-inflammatory effect and protection of SSTF to learning and memory damage induced by Aβin rat.Method:1 The serum preparation, detection of factors in serum, hippocampus, cortex:The blood was collected from heart in rat. The activities of SOD, GSH-Px and CAT, and the content of MDA were detected in serum and hippocampus and cortex.2 detection of TNF-αIL-1βin serum, GFAP expression in hippocampus.Result:1 SOD, GSH-Px, CAT and MDA in rat serumThe activities of SOD(80.88), GSH-Px(1613.27) and CAT(8.34) in serum were significantly decreased in model compared with those of control group(99.70,1878.86,13.76). Those of SSTF-M, SSTF-H and VE groups were significantly increased compared with that of model group.The content of MDA (12.38) in serum was significantly increased in model compared with that of control group (6.40). The content of MDA in SSTF-M(9.45), SSTF-H(7.51) and VE(9.30) groups were significantly decreased compared with that of model group.2 SOD, GSH-Px and MDA in rat hippocampusThe activities of SOD(72.87) and GSH-Px(135.16) in hippocampus were significantly decreased in model group compared with those of control group (100.24,170.74). Compared with that of model group, the activities of SOD were significantly increased in SSTF-M(94.58), SSTF-H(96.77) and VE(91.91) and the activity of GSH-Px (168.67) was significantly increased in SSTF-H.The content of MDA in hippocampus was significantly increased in model(8.39) compared with that of control group(4.96), compared with that of model group, the content of MDA was significantly decreased in SSTF-M(6.31), SSTF-H(5.30) and VE group(6.35) groups.3 SOD, GSH-Px and MDA in rat cortex The activities of SOD(69.99) and GSH-Px(68.77) in cortex were significantly decreased in model group compared with those of control(103.35, 88.09)group. Compared with that of model group, the activities of SOD were significantly increased in SSTF-M(95.17), SSTF-H(99.38) and VE(93.45) and the activity of GSH-Px was significantly increased in SSTF-H (85.83).The content of MDA in cortex was significantly increased in model(10.06) group compared with that of control(5.53) group. Compared with that of model group, the content of MDA was significantly decreased in SSTF-M(8.05), SSTF-H(6.23) and VE (8.01) groups.4 Quantity of glial cell in hippocampus CA1 region was significantly increased in model group compared with that of control group, there was no obvious difference among model and SSTF-M, SSTF-H groups. The contents of TNF-α,IL-1βin serum were significantly increased in model group compared with those of control group. Compared with that of model group, the contents of TNF-αIL-1βin serum were not altered in SSTF-M,SSTF-H groups.Conclusion:1 Injection of Aβin hippocampus could decrease the activity of SOD,GSH-Px and CAT in serum, SOD,GSH-Px activity in hippocampus and cortex, increase the production of oxidative products, induce neuron damage. Administration of SSTF could attenuate decreased enzyme activity induced by Aβ, decrease the production of oxidative products, attenuate neuron damage, protect neuron. This effect showed dose-dependent.2 Injection of AP in hippocampus could increase synthesis of TNF-a, IL-1βin serum, Administration of SSTF had no effect on this change.Part three Anti-apoptosis effect of SSTF to learning and memory damage induced by Aβin ratObjective To explore the relationship between anti-apoptosis effect of SSTF and its protection to learning and memory damage induced by Aβin ratMethod:1 The cell apoptosis was detected with TUNEL method. 2 The expression of bax,bcl-2, caspase-3,cyt-c in hippocampus and cortex, and IOD of positive cell was respectively detected with immunohistochemistry method.3 The expression of bax, bcl-2, caspase-3 and cyt-c in hippocampus was respectively measured with Western blotting. Calculate the ratio of bax, bcl-2, caspase-3, cyt-c to P-actin respectively.Result:1 The detection of TUNEL showed that brown appeared in nucleus of apoptosis neuron, more positive cell appeared in hippocampus CA1 region and cortex of model than in control. IOD in CA1 region(40.41) and cortex(27.73) were significantly increased in model compared with that of control(7.77, 1.34). IOD was significantly decreased in SSTF-M and SSTF-H compared with that of model.2 Immunohistochemistry:2.1 Expression of bax appeared in plasmid in brown, positive cell of CA1 region and cortex were increased in model compared with those of control. Positive cell was significantly decreased in SSTF-M, SSTF-H compared with that of model. IOD of CA1 region(144.51) and cortex(57.83) in model were significantly higher than those of control(20.78,13.43). Those in SSTF-M and SSTF-H were significantly decreased compared with that of model.2.2 Bcl-2 expression appeared in plasmid in brown, positive cell in CA1 region and cortex were significantly decreased in model compared with those of control. IOD of CA1 region(21.45) and cortex(2.67) in model were significantly lower than those in control(115.24,31.56). Those in SSTF-M and SSTF-H were significantly increased compared with that of model.2.3 Caspase-3 expression appeared both in plasmid and nuclear. IOD of CA1 region (126.28) and cortex(18.45) in model were significantly higher than those in control(22.47,3.27). Those in SSTF-M and SSTF-H were significantly decreased compared with that of model.2.4 More cyt-c expression appeared in plasmid, a few appeared in nuclear. IOD of CA1 region(124.79) and cortex(16.57) in model were significantly higher than those in control(25.13,2.86). IOD in SSTF-M and SSTF-H were significantly decreased compared with that of model.3 western blotting:The bax/p-actin(0.2678), caspase-3/β-actin(0.16),cyt-c/p-actin (1.2491) in hippocampus were significantly higher in model than those of control(0.1027,0.0388,0.6053). Those of SSTF-M and SSTF-H were significantly decreased compared with that of model.The bcl-2/p-actin in model(0.2354) was significantly lower than that in control(0.4131), that in SSTF-H(0.2964) was significantly increased compared with that of model.Conclusion:Injection of Aβin hippocampus could promote the expression of pro-apoptosis gene such as bax,caspase-3,cyt-c, inhibit the expression of anti-apoptosis gene bcl-2, induce neuron apoptosis in hippocampus and cortex. Administration of SSTF could decrease expression of bax,caspase-3,cyt-c, and increase expression of bcl-2, attenuate neuron apoptosis induced by Aβ.Conclusion:1 The decreasing of learning and memory induced by injection Aβin hippocampus in rat was related to decrease activity of anti-oxidant enzymes, increase oxidative products, neuron damage induced by AP; also was related to release of pro-inflammatory factors such as TNF-a, IL-1β. The increasing of oxidative products and release of pro-inflammatory factor could promote expression of pro-apoptosis gene bax,caspase-3,cyt-c, and inhibit expression of anti-apoptosis gene bcl-2, and deteriorate neuron damage.2 SSTF has a beneficial protection against the learning and memory injury induced by Aβ, and showed dose-dependent. The mechanism might be promoting activities of anti-oxidant enzymes, decreasing production of oxidative products, decreasing expression of pro-apoptosis gene, increasing expression of anti-apoptosis gene.