| Objective: In this study,a rat model of Cognitive impairment after subarachnoid hemorrhage(SAH CI)was established.To observe the effects of "Olfactory three-needle" on behavior,hippocampal neuron morphology,Wnt/β-catenin molecular signaling pathwayrelated protein expression,and hippocampal neural stem cells(NSCs)proliferation in rats with SAH CI.To clarify the positive intervention effect of "Olfactory three-needle" on the proliferation of hippocampal NSCs by activating the Wnt/β-catenin signaling pathway in the brain of SAH CI rats,to reveal the internal mechanism of "Olfactory three-needle" in slowing down SAH CI.Methods: Sixty SPF male Sprague-Dawley rats,aged 8 weeks,weighing 200g±20g,were randomly divided into the blank group(n = 10)and the model group(n = 50)after normal feeding for one week,and the SAH model was prepared by injecting blood into the optic chiasm of the 50 rats in the model group.Forty-five rats with cognitive impairment after modeling were randomly divided into the SAH model group,"Olfactory three-needle" group,"Olfactory three-needle" +DKK1 inhibitor group,piracetam group,and sham acupuncture group,with 9 rats in each group.In the olfactory three-acupuncture group and the olfactory three-acupuncture plus DKK1 inhibitor group,EA was applied to bilateral Yingxiang points and Yintang points,once a day for 14 days.The Piracetam group was given piracetam tablets aqueous solution by gavage,once a day for 14 days.In the sham acupuncture group,the Yintang point and Yingxiang point were left about 0.5 cm backward,respectively,and the needles were retained according to the time of the experimental group.At the end of the last treatment,the modified Garcia scale,Morris water maze test,and rotary-rod fatigue test were used to detect the therapeutic effect of the rats in each group.After the end of the treatment,the samples were taken,and the morphological changes of the hippocampus were observed by hematoxylin-eosin(HE)staining.The protein expressions of Wnt1,β-catenin,LMNB1,and GSK-3β in the hippocampus were detected by Western blot(WB).In addition,the proliferation of NSCs in rat hippocampal neurons was observed by immunohistochemistry and immunofluorescence.Results: 1.The effect of "Olfactory three-needle" on learning,memory,and other behaviors in the rats with SAH CI: after the model was established,compared with the blank group,the cognitive impairment of the SAH CI model group was serious,and the learning and memory and motor coordination were significantly impaired(P<0.01).Compared with the SAH CI model group,the symptoms of cognitive impairment in the "Olfactory three-needle" group and the piracetam group were significantly improved(P<0.01,P<0.01).The results of the Morris water maze test and rotarod fatigue test were consistent,indicating that the credibility of this study was good.The results of the two behavioral tests showed that "Olfactory three-needle" can effectively treat CI after SAH,and improve the symptoms of learning spatial memory ability and motor coordination disorder in rats.2.The effect of "Olfactory three-needle" on the morphology and structure of hippocampal neurons in the brain of rats with SAH CI: the results of HE staining showed that the cytoplasm and inclusions of hippocampal DG area cells in the blank group were neatly arranged and the contour was regular,and no neuronal atrophy or volume reduction was observed.In the model group,the "Olfactory three-needle" +DKK1 inhibitor group,and the sham acupuncture group,there were large necrotic cavities in the neuronal area,necrotic tissue edema,and atrophy,irregular cell morphology,unclear edge,accompanied by pyramidal cell necrosis,and varying degrees of cell fragmentation in the surrounding area.Compared with the model group,the "Olfactory three-needle" group and the piracetam group had improved cell morphology,some pyramidal cell necrosis,reduced cell laxity,and reduced vascular dilatation and congestion in the injury site.3.To investigate the effect of "olfactory three-needle" on the expression of key proteins in the Wnt/β-catenin signaling pathway in the hippocampus of rats with CI after SAH: Compared with the blank group,the protein levels of Wnt1,β-catenin,and LMNB1 in the hippocampus of the SAH CI model group were decreased(P<0.01,increased GSK-3β protein(P<0.05).Compared with the model group,the expressions of Wnt1,β-catenin,and LMNB1 proteins in the hippocampus of the "Olfactory three-needle" group and the piracetam group were significantly increased(P<0.05 or P<0.01)and GSK-3β protein(P<0.05).The results indicate that the "Olfactory three-needle" can effectively activate the Wnt/β-catenin signaling pathway,regulate the expression of its downstream molecules,and improve cognitive impairment.4.The effect of "Olfactory three-needle" on the proliferation of NSCs in the hippocampus of rats with CI: compared with the blank group,the number of Brd U-positive cells and Neu N-positive neurons in the hippocampal DG region of the model group was significantly decreased(P<0.01),which indicated that the proliferation ability of NSCs in the hippocampus of SAH CI rats was significantly reduced;Compared with the model group,the number of Brd U-positive and Neu N-positive neurons in the hippocampal DG region of the "Olfactory three-needle" group and piracetam group were significantly increased(P<0.01),which indicated that the proliferation of NSCs in the hippocampus was enhanced after olfactory three-needle treatment,and there was no difference compared with piracetam group(P<0.05).The results of Brd U immunohistochemistry and Neu N immunofluorescence in the hippocampus of each group were also consistent with the results of WB,which confirmed the stability and credibility of the "Olfactory three-needle" in promoting the proliferation of hippocampal NSCs in the rats with SAH CI.Conclusion: "Olfactory three-needle" intervention can improve the learning,spatial memory,and motor coordination ability of SAH CI rats by regulating the Wnt/β-catenin signaling pathway,promote the repair of damaged hippocampal neurons in rats,and significantly improve the cognitive ability of model rats.Its mechanism may be that it can up-regulate the expression of Wnt1,β-catenin,and LMNB1 proteins in the hippocampus,inhibit the expression of GSK-3β protein,promote the proliferation of hippocampal NSCs,reduce the damage of hippocampal neurons,and thus improve cognitive impairment. |
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