| ObjectiveIn this study,a rat model of chronic cerebral ischemic injury was established by permanent ligation of bilateral common carotid arteries.The effects of RQKL on spatial learning and memory ability and hippocampal neuron morphology in model rats were observed.Histomorphology was evaluated for initial efficacy.On this basis,the effects of RQKL on the expression of neurotrophic factor BDNF and receptor TrkB,nerve growth factor NGF and neurotrophic factor NT-3 in hippocampal CA1 area of model rats were studied in order to explain partially mechanism of action of cognitive disorders caused by cerebral ischemia with the treatment of RQKL.Method(1)A rat model of chronic cerebral ischemia was prepared by permanently ligating the bilateral common carotid arteries in experimental rats.The experimental animals were divided into Sham group,Model group,Donepezi group,and RQKL group.The rats were given a continuous administration of the drug for 60 days after modeling.Through the Morris water maze behavioral experiment,the navigation and space exploration tests were carried out for each group,and the experiment was carried out for 5 days.After the end of the water maze test,some experimental rats in each group were randomly selected for perfusion fixation,and hippocampal paraffin sections were prepared for HE staining.Morphological changes of hippocampal neurons were observed under light microscope.(2)The expressions of brain-derived neurotrophic factor BDNF and its receptor TrkB in hippocampal CA1 region were determined by immunohistochemistry and Westen-blotting.The expression of BDNF mRNA and TrkB mRNA were detected by Real Time PCR.We analyze the effect of RQKL on the expression of BDNF and TrkB.(3)Immunohistochemistry and enzyme-linked immunosorbent assay were used to determine the expression of neurotrophic factor NT-3 and NGF protein in hippocampal CA1 region.The expression of NGF mRNA and NT-3 mRNA was measured by Real Time PCR..We will analyze the effect of RQKL on the expression of NGF and NT-3.Result(1)In the chronic cerebral ischemia model,some behavioral and histomorphological changes were observed after permanent bilateral common carotid artery ligation.In the water maze test,rats with chronic cerebral ischemia showed significant learning and memory impairment.Compared with the Sham group,the decline tendency of the escape latency of the Model group was relatively flat,and the escape latency was significantly prolonged(P<0.05).The number of crossing the platform and the number of crossing the original platform quadrants in the Model group were significantly reduced(P<0.01),statistically significant;HE staining showed that the number of neurons in the hippocampal CA1 area of the Model group was significantly lower than that of the Sham group,the pyramidal cells were loosely arranged,the cell bodies were shrunk,the nucleus was pyknotic or dissolved,the cell spacing became larger,and a small number of nerves The cells showed edema and showed some obvious ischemic pathological changes.(2)After 60 days of continuous gavage of RQKL drugs,the results of water maze positioning experiments showed that compared with the Model group,the decline latency of the rats in the RQKL group was significantly decreased,and the escape latency was significantly shortened(P<0.05).The results of space exploration experiments showed that compared with the Model group,the number of crossing the platform and the number of quadrants crossing the original platform in the RQKL group were significantly increased(P<0.05),which was statistically significant.By HE staining,the number of neurons in the RQKL group was increased compared with the Model group,the loss of nerve cells was reduced,the cells were arranged in an orderly manner,the cell boundaries were clear,the pyknosis phenomenon was reduced,and the number and morphology of the cells were close to the Sham group.(3)The protein levels of brain-derived neurotrophic factor BDNF and its receptor TrkB in hippocampal CA1 region and the expression of BDNFmRNA and TrkB mRNA were significantly changed in the hippocampal CA1 area.Immunohistochemical staining showed that compared with the Model group,the brown-yellow expression of BDNF and TrkB in the hippocampal CA1 area of the RQKL group was significantly increased(0.48±0.06,P<0.05;0.39±0.05,P<0.05).The results of mmunoblot analysis showed that compared with the Model group,the BDNF and TrkB protein contents in the RQKL group were significantly increased(0.49±0.07,P<0.05;0.41±0.09,P<0.01,respectively).The results of RT-PCR showed that the expression of BDNF mRNA and TrkB mRNA in RQKL group was significantly higher than that in Model group(0.79±0.10,P<0.05;0.71±0.04,P<0.05).(4)Under the administration of RQKL,the expression of neurotrophic factor NT-3,nerve growth factor NGF protein and NT-3mRNA?NGFmRNA in hippocampal CA1 area were significantly changed.The results of IHC showed that compared with the Model group,the expression of neurotrophic factor NT-3 and NGF in the hippocampal CA1 region of RQKL group increased(0.58±0.04,P<0.01;0.33±0.04,P<0.05).The results of enzyme-linked immunosorbent assay showed that compared with the Model group,the contents of NT-3and NGF in the hippocampus of RQKL group increased significantly(152.39±16.34,P<0.01;119.30±12.07,P<0.05).The results of Real Time PCR showed that compared with the Model group,the expression levels of NT-3 mRNA and NGF mRNA in RQKL group were significantly increased(1.35±0.11,P<0.01;0.84±0.08 P<0.05,respectively).Conclusion(1)RQKL can improve the ability of spatial learning and memory in rats with chronic cerebral ischemia to a certain extent,and reduce the damage of rat hippocampal neurons induced by chronic cerebral ischemia.(2)Purified Qingkailing can up-regulate the protein levels and mRNA expression of brain-derived neurotrophic factor BDNF and its receptor TrkB in hippocampal CAI region,indicating that it promotes the effective binding of BDNF and its receptor TrkB,thereby exerting protective effects on neurons.It may be one of the ways to purify Qingkailing against chronic cerebral ischemic inj ury.(3)RQKL can up-regulate the expression of neurotrophic factor NT-3,NGF protein and mRNA in hippocampal CA1 region,and play a protective role on neurons to a certain extent.It may be one of the ways to purify Qingkailing against chronic cerebral ischemic injury. |
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