Effects Of Glycosides Of Cistanche On Synaptic Morphological Plasticity in Senescence-accelerated Mice
Mar 11, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
LIAN Juanqi1, WANG Lu2, ZHAO Fujun3, LIN Shuai3, YAN Xusheng4, JIA Jianxin4, CAI Zhiping4
(1. Grade 2014 of Graduate School of Baotou Medical College, Baotou 014060, China)
(2. Class 12 of Grade 2015 Clinical Medicine in Baotou Medical College)
(3. Class 1 of Grade 2013 Forensic Medicine in Baotou Medical College)
(4. Department of Human Anatomy of Baotou Medical College)
ABSTRACT
Objective: To investigate the effect of glycosides of cistanche (GCs) on the synaptic plasticity of hippocampal neurons in senescence-accelerated mice prone 8 (SAMP8). Methods: 40 mice were randomly divided into control group, vehicle group, low-dose group (administered GCs 2.5 mg / kg ·d), middle-dose group (administered GCs 5.0 mg / kg? d) and high dose group (administered GCs 10. 0 mg / kg · d). The mice in the control group were not given any intervention; those in administered groups and the vehicle group underwent intraperitoneal administration (GCs / saline) or saline for 30 d. The number of normal pyramidal neurons in the hippocampal CA1 region was counted by Nissl staining; the number of dendritic spines in synapses was counted by Golgi staining, and the protein expression of PSD-95 was quantified by immunohistochemistry． Results: Compared with those in the control group, GCs significantly increased the number of normal pyramidal neurons in the hippocampal CA1 region (P <0.05) and increased the number of dendritic spines in synapses (P <0.05). The expression levels of PSD-95 in administered groups were significantly higher than that in the control group (P <0.05), and the effect in the middle dose group was the most significant. Conclusion: GCs can im- prove the survival rate of hippocampal pyramidal cells and improve synaptic plasticity．
KEYWORDS: Glycosides of cistanche (GCs); Senescence accelerated mouse; Dendritic spine; PSD－95
With the aging of the social population becoming more and more serious, the prevalence of Alzheimer's disease (AD) has gradually increased, which has brought a heavy burden to patients' families and society
[1]. The main clinical manifestations of AD are cognitive impairment and memory impairment, and the main case features are senile plaques formed by extracellular amyloid deposits and intracellular neurofibrillary tangles[2]. The pathogenesis of AD is still inconclusive. The pathological changes of synapses are considered to be the neurobiological basis of AD memory and cognitive dysfunction. Abnormal changes in synapses may be the core link in the pathogenesis of AD. Senescence accelerated mouse (SAM) is an inbred aging model mouse. Its P8 subline has uniform and stable aging pathological characteristics, which is manifested by a progressive decline in learning and memory and recognition with the increase of months. Known dysfunction and pathological manifestations are similar to AD patients, so SAMP8 mice are an ideal animal model for studying the mechanism of aging-related learning and memory impairment.
This article intends to analyze the cell survival rate, measure dendritic spine density, and detect post-synaptic compacts to explore the possible mechanism of GCs (glycosides of cistanche)affecting the synaptic morphological plasticity of hippocampal neurons in rapidly aging mice, with a view to treating AD-related cistanche glycosides (GCs) The spatial learning and memory impairment provides theoretical and experimental basis.
1 Material and methods
1.1 Experimental animals and grouping 40 male 10-month-old SAMP8 mice (purchased from Tianjin University of Traditional Chinese Medicine), reared in a clean environment, ambient temperature (24.0 ± 1.0) ℃, humidity (50.0 ± 5.0)%, free food and water. The animals were raised to 11 months of age to start the experiment. The animals were randomly divided into 5 groups (8 animals/group): blank control (control) group, solvent control (vehicle) group, cistanche total glycosides (GCs) low-dose group [2.5 mg / (kg ·d)], Medium dose group [5. 0 mg / (kg · d)], high dose group [10.0 mg / (kg · d)]. Animals in the control group were not given any intervention. The animals in the low-dose group were given GCs 2.5 mg / (kg ·d), the animals in the middle-dose group were given GCs 5.0 mg / (kg ·d), and the animals in the high-dose group were given GCs. 0 mg / (kg ·d), animals in the vehicle group were given the same amount of normal saline. Continuous intraperitoneal injection of drugs or physiological saline for 30 days, 0.5 mL/d.
1.2 Specimen preparation and processing The animals were sacrificed 24 hours after the last administration, anesthetized, and the neck was sacrificed. Cut the thoracic cavity to expose the heart, cut the right atrial appendage to let the blood out, and insert the perfusion needle into the left ventricle. After 100 mL of normal saline was infused, 4% paraformaldehyde was continued to be infused until the mice’s limbs were stiff. Take the brain and place it in 4% paraformaldehyde for more than 24 hours.
1.3 Drugs and reagents GCs(glycosides of cistanche) were purchased from China Academy of Traditional Chinese Medicine, postsynaptic density －95, PSD －95 were purchased from Epitomic, Coomassie Brilliant Blue CBBG 250 kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. Engineering Institute, DAB immunohistochemistry kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Company.
1.4 Nissl staining and cell count 4% paraformaldehyde fixation of brain tissue for more than 24 hours, trimming and dehydration with gradient alcohol, transparent xylene, wax immersion, conventional paraffin embedding, 5 μm thickness serial section. Cut slices and dewax to water, dye with 1% toluidine blue at 37 ℃ for 60 min, wash for 5 min × 3 times, differentiate with 95% alcohol, 100% alcohol Dehydrate, transparent xylene, mount with neutral gum, observe under the microscope. The CA1 area of the same hippocampal section of each group was randomly selected from 3 observation fields (×100), the number of normal pyramidal cells within 1 mm was counted, and the average was taken for analysis.
1.5 Modified Golgi staining Put the repaired brain tissue in Golgi-Cox staining solution, protect from light for 14 days, and change the solution every 2 days; place the brain tissue in 30% sucrose, and place it in a refrigerator at 4 ℃. The tissue can be sectioned after sinking to the bottom; vibrating microtome for sectioning and staining; fixer solution for development; the drying of the mounting plate, observation, and counting under the microscope. Observe the changes in the dendritic spine of the neuronal apex of the hippocampus CA1 and CA3 of each group of mice, and calculate the density of the dendritic spine.
1.6 The immunohistochemical sections were deparaffinized to water, incubated with 3% H2O2 for 10 minutes to eliminate endogenous peroxidase activity, washed with distilled water for 5 minutes × 3 times, repaired by high-pressure microwave, washed with PBS for 5 minutes × 3 times, Goat serum was blocked for 30 min at room temperature, rabbit anti-mouse PSD-95 primary antibody was added dropwise, overnight at 4 ℃, washed with PBS for 5 min × 3 times, biotin-labeled secondary antibody was added dropwise, incubated at 37 ℃ for 2 h, washed with PBS for 5 min × 3 times, drop the horseradish peroxidase-labeled streptavidin, wash with PBS for 5 min × 3 times, wash with DAB for 2 min, counterstain with hematoxylin, dehydrated with gradient alcohol, transparent with xylene, and mount. 1.7 Image processing and statistical analysis. The same hippocampal cross-sections of each group were selected for observation. Three fields of view (×100) were randomly selected in the CA1 area to count the number of normal pyramidal cells within 1 mm, and the average was analyzed. The results of immunohistochemistry were measured by IPP 7.0 image analysis software. The experimental data were analyzed by SPSS 17.0 software, and one-way ANOVA was used for multi-sample means pairwise comparison. P<0.05 was considered statistically significant.
Anti-Alzheimer's disease
2 results
2.1 The effect of the total glycosides of cistanche on the count of normal pyramidal cells in the hippocampus of SAMP8 mice Nissl staining The results of Nissl staining showed that the number of neurons in the CA1 area of the control group decreased, the arrangement was disordered, and most of the cells were abnormal in morphology and lysis and necrosis. The morphology of the cells in the GCs(glycosides of cistanche) group was relatively clear and complete, with a large number of protrusions visible, the Nissl body was purple, the nucleoli were deeply stained, clear, and centered, and the nuclei were not stained. The cell morphology of the vehicle group is similar to that of the control group. The cell count showed that the number of normal pyramidal neurons in the CA1 area of the administration group increased (P <0.05); the difference between the medium and high dose groups was not statistically significant (P>0.05), the number of normal pyramidal neurons To a certain extent, it shows an upward trend with increasing dose. There was no significant difference in the number of normal pyramidal cells between the vehicle group and the control group (P>0.05). See Table 1.
Table 1 Effect of Cistanche Glycosides on SAMP8 Mouse Hippocampal Nis Staining, Normal Pyramidal Cell Count and Dendritic Spines Number (pcs)
a is compared with the control group t = 0.193, P = 0.582; b is compared with the control group t = 13.621, P = 0.008; c is compared with the control group t = 21.798 , P = 0.005; d is compared with control group t = 18.326, P = 0.006; e is compared with control group t = 0.244, P = 0.463; f is compared with control group Compared with t = 10.903, P = 0.010; g is compared with the control group t = 21.817, P = 0.005; h is compared with the control group t = 15.572, P=0. 007.
2.2 The effect of the total glycosides of cistanche on the density of dendritic spines in SAMP8 mice Golgi staining showed that the number of dendritic spines in the control group decreased, and the density decreased. After giving animals GCs, the number and density of dendritic spines increased, and a large number of protrusions were seen (P = 0.048). There was no statistically significant difference between the middle-dose group and the high-dose group (P = 0.853). The number and density of the dendritic spine The degree shows an upward trend with the increase of dose. vehicle group and control
The number and density of dendritic spines in the group have no statistical significance (P = 0.764). See Table 1.
2.3 The effect of Cistanche glycosides on the expression of PSD-95 in SAMP8 mice The immunohistochemical qualitative detection of PSD-95 showed that the control group had less expression of PSD-95, the cell body staining was lighter, and the positive expression rate was lower. After supplementation of GCs in vitro, the cell body stained deeper, and the expression and positive rate of PSD-95 increased (t = 5.751, P <0.05). There was no significant difference between the middle-dose group and the high-dose group (t = 1.077, P>0.05). To a certain extent, the expression of PSD-95 showed an upward trend with increasing doses. vehicle
There was no statistically significant difference in PSD-95 expression between the control group and the control group (t = 0.585, P>0.05).
3 Discussion
PSD-95 is the main component of postsynaptic and is related to a variety of synaptic functions, involving ion channels, synaptic activity, intracellular signal transduction pathways, etc. [3]. PSD-95 clusters related proteins in the NMDA receptor signaling pathway to form a receptor-signaling molecule-regulatory molecule-targeting molecule complex [4], which plays a key role in the signal transduction pathway. NMDA receptors are involved in the induction of long-term potentiation (LTP) in the CA1 region of the hippocampus and the process of learning and memory [5]. PSD-95 plays a key regulatory role in this process, and its protein content can be used as Another molecular marker for measuring synaptic plasticity. In our previous studies, we have proved that GCs(glycosides of cistanche) can improve learning and cognitive impairment in SAMP8 mice, but we have not clarified its possible cell morphological mechanism. This experiment aims to study its possible cell morphological mechanism.
In this experiment, the improvement of pyramidal cell survival rate and synaptic plasticity in the GCs(glycosides of cistanche) group was better than that of the vehicle group and the control group. It can be seen that GCs improved the morphology of pyramidal cells more significantly. The experimental results show that higher doses of GCs can exert a better effect. From the comparison between the middle dose group and the high dose group, it can be seen that the effect does not change significantly when the dose reaches a certain concentration. Therefore, in order to avoid waste and side effects of GCs when using GCs, the most appropriate concentration should be selected [6].
Previous studies have shown that GCs(glycosides of cistanche) can improve the learning and cognitive impairment of SAMP8 mice by improving the survival rate of pyramidal cells [7]. There are also data indicating that GCs may improve neurodegenerative diseases by promoting the proliferation and differentiation of neural stem cells [8-10]. The author believes that the mechanism for GCs to improve AD may be achieved by improving the plasticity of synaptic morphology and maintaining the number of pyramidal cells, which lays the foundation for the ultimate use of GCs in the prevention and treatment of clinical AD.
References
[1] Chen Juluo, Chen Xingui, Hu Panpan, etc. Alzheimer's disease patients use functions Preliminary research [J]. Chinese Journal of Behavioral Medicine and Brain Science, 2013, 22 (10): 889-891.
[2] Jin Chunhui, Liu Xiaowei, Yuan Jianmin, et al. Sorting Receptor 1 Gene Single Nucleoside Correlation between acid polymorphism and sporadic Alzheimer's disease［J］. Chinese Journal of Behavioral Medicine and Brain Science, 2014, 23 (5): 427-429.
[3] Schliebs R, Arendt T. The significance of the cholinergic system in the brain during aging and in Alzheimer's disease［J］. Neural Transm, 2006, 113 (11): 1625-1644.
[4] Soriano FX. Specific targeting of pro-death NMDA receptor signals with differing reliance on the NR2B PDZ ligand［J］． J Neurosci, 2008, 28 (42): 10696-10710.
[5] Selkoe DJ. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior ［J］． Behav Brain Res, 2008, 192 (1): 106 -113.
[6] Huang Zonghui, Chen Guanmin, Zhao Kangtao, et al. Study on the toxicity of Cistanche ［J］. China Journal of Hygiene Inspection, 2014, 24 (8): 1098 -1100.
[7] Jia Jianxin, Song Wei, Yan Xusheng, et al. The total glycosides of cistanche have an effect on SAMP8 mice. The influence and mechanism of inter-learning and memory［J］. Journal of Baotou Medical College, 2014, 30 (6): 6-8.
[8] Xiao Q, Yan P, Ma X, et al. Enhancing astrocytic lysosome biogenesis facilitates Aβ clearance and attenuates amyloid plaque pathogenesis［J］． J Neurosci, 2014, 34 (29): 9607-9620.
[9] Li QJ, Tang YM, Liu J, et al. Treatment of Parkinson's disease with C172 neural stem cells over-expressing NURR1 with a recombined republic－deficit adenovirus containing the NURR1 gene［J］. Synapse, 2007, 61 (12): 971-977.
[10] Qin Wei, Wei Wei, Xu Qian, et al. Cistanche on the proliferation of C172 neural stem cells The impact of [J]. Strait Pharmacy, 2015, 27 (12): 263-265.
