Supported by National Natural Science Foundation of China (81673856, 81573865); China Postdoctoral Science Foundation (2016M592319, 2017T100542); Youth Project of Hubei University of Traditional Chinese Medicine (ZhongYiXiaoZi2015182); PhD Research Foundation of Hubei University of Traditional Chinese Medicine (ZhongYiDang Zi201425).

1 Introduction

The senile dementia is a neurodegenerative disease with aging as the main cause, and AD (Alzheimer’s Disease) is the main type of senile dementia with pathological manifestations of the senile plaques formed by the aggregation of extracellular β-amyloid in brain tissue (Aβ), and the neurofibrillary tangles and neuronal loss formed by the aggregation of intracellular hyperphosphorylation of tau protein[1]. With the intensification of the process of aging in the society, the incidence of AD patients is also gradually increasing, the current number of patients with AD is up to 5 000 000, accounting for 25% of the number of patients worldwide, and the incidence of AD reaches as high as 7.5% for the people aged more than 65 years[2]. Although the countries all over the world have spent a lot of time, manpower and financial resources on exploring the exact pathogenesis and effective treatment means, it has had little success.

由上述西部矿业企业的财务指标分析中可知，虽然企业近五年的长期偿债能力一直处于良好水平，且企业的发展状况较好，但企业的营运能力和盈利能力总体来说是处于下降阶段的，短期偿债能力总体较弱，企业在财务方面的竞争力方面优势较小、营运能力较弱，且企业的流动比率与速动比率也较小，资金流动性不强，导致资金成本增加。

Chinese medicine believes that there are pathological features of asthenia in origin and asthenia in superficiality in the Alzheimer’s Disease, with deficiency of the kidney as the root cause and phlegm obstruction and blood stasis as symptoms, so the method of invigorating the kidney, resolving phlegm and promoting blood circulation by removing blood stasis is commonly used in clinical prevention and treatment of AD. Notoginseng is the Chinese medicine used to stop bleeding, promote blood circulation by removing blood stasis and tonify the blood, in line with the theory of traditional Chinese medicine treatment of Alzheimer’s disease by activating blood and resolving stasis. The notoginseng contains notoginsenosides, flavonoids, polysaccharides, dencichines, amino acids and other substances, and notoginsenosides are the main active constituent of notoginseng. The arasaponins contain various monomer components that have been isolated and identified, of which Rg1 is believed to be about 20% of notoginseng roots. Modern studies have found that notoginseng can improve the memory and cognitive function, resist the free radical toxicity impairment, inhibit the apoptosis regulating genes, and have a certain therapeutic effect on AD[3]. However, what on earth are the constituents to function? What molecular mechanisms are involved? These still need further study.

如图1所示，为了尽可能提高M2e的蛋白量，采用3个M2e序列串联形成3M2e；同时为了提高小多肽段（M2e）的抗原性，在3M2e前加接一个鞭毛蛋白基因（FljB基因），合成出Flagellin-3M2e的DNA片段。 目的基因Flagellin-3M2e被构建于表达载体pET28a相应位置（EcoRⅠ-XhoⅠ），经内切酶鉴定（图1）和基因测序确定序列正确。

2 Materials and methods

2.2.1 Stereotaxis and administration. Before the experiment, one week of adaptive feeding was conducted for the animals, and the Morris water maze tested was done to eliminate the unqualified rats. During the rat stereotaxis, it was first injected intraperitoneally with 6% chloral hydrate for anesthesia (400 mg/kg), the routine disinfection was carried out, and the skin was preserved and fixed on the rat brain stereotaxic instrument. Referring to Rat Brain Stereotaxic Mapping prepared by Fanklin and Paxinos, the left lateral ventricle was positioned[4]. The selected sites were 0.8 mm behind the rat bregma, and 3.5 mm on the left side. The small hole with the diameter of about 1 mm was drilled in this site, and the microinjector was used to suction 2 μL of Aβ25-35 liquid to be slowly injected into the part 4.0 mm deep in the hole, namely left ventricle. After injection, the needle was retained in the body, and after taking out the needle, the medical gelatin sponge was used to fill the injection aperture, and the skin incision was conventionally sutured and disinfected. The normal group was injected with normal saline. One week later, the Morris water maze test was used to screen the successfully modeled rats. After 24 h, the low, middle, high doses of notoginsenoside Rg1 (25, 50, 100 mg/kg, respectively) were intragastrically administered, and the normal group and model group were orally fed with normal saline for 4 weeks, 2 times/d. After 4 weeks, the rats were killed after using the Morris water maze to test the spatial learning and memory of each group of rats, and the left hippocampal tissue was taken and stored at -80℃ or the brain tissue was taken by perfusion for later use.

2.2.2 Testing of rats’ spatial learning and memory capacity by Morris water maze. The rats in each group were tested after 4 weeks of intragastric administration, and the 7-d water maze training test was conducted on each group of rats. The water maze was divided into two parts: a cylindrical pool and an automatic video analysis system. The pool was divided into four quadrants, which were called the first, second, third and fourth quadrants, respectively. The water was 15 cm deep, the water temperature was controlled to be (22.0 ± 0.5)℃, and a cylindrical platform was placed in the middle of the third quadrant (wrapping with the black plastic bag outside). The pool water surface was 1-2 cm above the platform surface, an appropriate amount of ink was added to the water so that the platform was invisible, and the same platform position was maintained in the experiment. Each experiment time was set to be 60 s, and the rats facing the wall were put into the pool from the first, second, third and fourth quadrants, respectively. The movement trajectory of rats was recorded simultaneously with a video camera above the pool, and the time of rats finding the rats and staying for 5 s was the incubation period. If the rats failed to find the platform, the experimenter would guide them to the platform and stay 30 s to adapt to the surroundings, followed by the next round of experiments. The training was conducted 4 times a day, one time for each quadrant. On the 7th day, the rats were placed from any quadrant after the platform was removed, the first platform crossing time, namely the incubation period, was tested, and the frequency of platform crossing was observed at the same time. The experimental time was fixed at 13: 00-20: 00 every day.

日前，云南省金沙江中游库区航运基础设施综合建设一期工程奉科码头、宝山码头、阿海上翻坝码头、阿海下翻坝码头、树底码头“五点一线”码头水工工程全部完工。投用后，将更好完善区域综合交通运输体系，服务四川、云南省沿江地区经济社会发展。

云南某露天采场属大型露天矿山，是一个生产多年的老矿山，具有剥采比大、剥离岩石量大、边坡高差大等特点，加上多年以来重采矿、轻剥离，造成矿山剥离欠账严重，工作面分散且落差较大，矿山开拓运输复杂。矿岩石运输均采用公路—汽车运输开拓，车辆拥挤，安全隐患较大。排土场设置较远，矿山岩石运输成本较高，按矿山目前外包承包价计算，排土场6.3 km的平均运距，每立方岩石采剥成本需要37.8元，其中运费就占到了25.20元左右，占采剥成本的66.7%，明显不合理。

2.2 Experimental methods

2.1 Materials

2.1.1 Reagents. Aβ25-35 was purchased from Sigma Company (USA), and after the Aβ25-35 peptide was dissolved in DMSO, the physiological saline was added for preparation into the solution with a concentration of 2.6 μg/μL. It was incubated in the dark incubator at 37℃ for one week, and placed in the refrigerator at 4℃ for later use. The notoginsenoside Rg1 was purchased from Beijing Zhong Ke Yi You Research Institute of Chemical Technology (standard 98%). The tau1, PT231 antibodies were produced by Chemicon Company (USA), and after subpackaging, they were stored in the refrigerator at -20℃. Before use, they were diluted with the antibody dilutions based on the ratio of 1∶1 000 for the Western blot detection, or they were diluted with the antibody dilutions based on the ratio of 1∶200 for the immunohistochemical detection.

1.4 观察指标 颈淋巴结清扫术时间：从切开皮肤到颈淋巴结清扫组织离体的时间，参照麻醉记录单；术中出血量：负压吸引瓶中的血量与术中使用纱布浸透的估计出血量的总和；术后引流量：术后24 h的引流量；术后住院天数；术后早期并发症，如皮下积液、创口渗漏等。

3.1 Effect of notoginsenoside Rg1 on the spatial learning and memory capacity of Aβ25-35-induced AD model rats After the intracerebroventricular injection with Aβ25-35 into the SD rats, the notoginsenoside Rg1 was used for intervention for four weeks. By detecting the spatial learning and memory capacity of animals with Morris water maze, it was found that the incubation period of the model group rats was (55.86 ± 4.31) s, (53.31 ± 3.01) s on the second and third day, respectively, significantly longer than the incubation period of the rats in other experimental groups (P<0.05), suggesting that the spatial learning and memory capacity of the model group rats significantly decreased; by comparing the incubation period (32.37±3.64)s and the platform crossing frequency (3.00 ±1.12) of the middle dose notoginsenoside Rg1 group with the incubation period (26.93±4.12)s and the platform crossing frequency (4.00±1.46) of the normal control group on the 7th day, the differences were not statistically significant (P>0.05); by comparing the incubation period (32.37±3.64)s and the platform crossing frequency (3.00 ±1.12) of the middle dose notoginsenoside Rg1 group with the incubation period (40.71±3.45 s) and the platform crossing frequency (1.00 ±0.26) of the model group on the 7th day, the differences were significant (P>0.05). It suggested that the middle dose notoginsenoside Rg1 could significantly reverse the spatial learning and memory capability impairment of the AD model rats; there were no significant changes in the incubation period of low dose notoginsenoside Rg1 group (38.75 ± 3.32)s and the incubation period of high dose notoginsenoside Rg1 group (37.08 ± 3.06)s, not significantly different from the model group (P>0.05). Meanwhile, there were also no significant differences (P>0.05) in the platform crossing frequency among the three groups, and there was a significant difference (P<0.05) in the incubation period and platform crossing frequency between the normal group and the low or high dose notoginsenoside Rg1 group. The above results suggested that the intracerebroventricular injection of Aβ25-35 into rats could induce the spatial learning and memory impairment of the AD rats, and the middle dose notoginsenoside Rg1 could effectively achieve reversal (no differences between the groups in the body weight of experimental animals during the experiment and the swimming speed in the water maze). The specific results are shown in Fig.1.

2.3 Statistical methods The SPSS16.0 software was used for statistical analysis, the measurement data were denoted by ±s, and the one-factor analysis of variance was used for comparison among more than two samples, and P<0.05 was considered statistically significant.

2.1.2 Experimental animals. 4-month old male SD rats (purchased from Hubei Provincial Center for Disease Control), weight of (280±20) g. The rats were randomly divided into the normal control group, model group, low dose group, middle dose group and high dose group, 10 rats per group.

2.3.4 Immunohistochemical method. Four animals in each group were perfused with paraformaldehyde and fixed before sectioning, and there was a need to try to pick the same section of brain slices in different groups on the 24-well plates. The right amount of PBS was used to slowly shake and wash on a shaker for 5 min×3. The membrane-rupturing solution was then added (30% H2O2 100 μL, Triton-100 50 μL, 1×PBS added to a constant volume of 10 mL) to for 15 min of rupture and 5 min×3 of 1×PBS shaking and washing. Then after 30 min of closure of 200-250 μL of 3% BSA-PBS solution, the primary antibody was added at 4℃ for 24-48 h of incubation, the primary antibody was recovered, and after 5 min×3 of shaking and washing with 1×PBS solution, the secondary and tertiary antibodies were sequentially incubated, followed by the DAB coloration. After placing the patch, the dehydration, permeabilization and mounting were conventionally conducted, and it was naturally dried for image acquisition.

3 Results

2.3.3 Immunoblotting method. The spare tissue was taken out and mixed with the homogenate based on the ratio of 1∶9 for homogenization. Buffer was added after ultrasonic disruption (Tris 2.42 g, HCl 1.5 mL, SDS 8.0 g, glycerin 40.0 mL, constant volume to 400 mL) to boil 10 min, then the protein concentration was measured by BCA method and the sample volume was calculated. The separating gel (lower gel) and stacking gel (upper gel) were formulated, the electrophoresis was conducted before the lanes were marked and the samples were loaded. During the electrophoresis to the bottom of gel, and the gel was removed and transferred to the NC membrane. After the completion of transmembrane, the non-specific sites were blocked, and the primary antibodies (tau1 of goat anti-mouse, PT231 of goat anti-rabbit) and the secondary antibodies (IgG fluorescent-labeled goat anti-mouse or goat anti-rabbit IRDyeTM, 800 channels). After Odyssey coloration, the gray value was detected.

Note: A. The Latency period results in the Morris water maze test of spatial learning and memory; B. The trajectory on day 1, 6, 7 of the Morris water maze test, n=8.

Fig.1 Effects of notoginsenoside Rg1 on the spatial learning and memory of the AD rats

3.2 Effect of notoginsenoside Rg1 on the hippocampal tau protein level in the Aβ25-35-induced AD model rats Neuronal fiber tangles formed by the accumulation of hyperphosphorylated tau protein in nerve cells are an important marker of the pathogenesis of AD. And it is closely related to the cognitive dysfunction of AD patients. The tau1 is a non-phosphorylated tau protein, and PT231 is one of the key sites of tau protein phosphorylation. In this study, we examined the level of PT231 and observed the effect of notoginsenoside Rg1 on the level of phosphorylated tau protein in the hippocampus of AD model rats induced by Aβ25-35. The results of Western blot indicated that the PT231 of the model group was significantly higher than that of the normal group (P<0.01), while the tau1 of the model group was significantly lower than that of the normal group (P<0.05). The notoginsenoside Rg1 could obviously reverse the increase of PT231 in AD model rats (P<0.05), but had no significant effect on non-phosphorylated tau protein tau1 (P>0.05). These results suggested that notoginsenoside Rg1 could reverse the increase of phosphorylated tau protein PT231 in AD model rats (Fig.2).

Note: A. Western blot results of PT231 and tau1 in each group; B. The statistical results of the relative gray value of Western blot bands in each group; compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05; n=4.

Fig.2 Western blot results of PT231 and tau1 in hippocampus after notoginsenoside Rg1 intervention in AD rat model

3.3 Effect of notoginsenoside Rg1 on the distribution of PT231 positive staining in the hippocampus of AD model rats induced by Aβ25-35 These results suggested that notoginsenoside Rg1 mainly influenced the phosphorylation level of tau protein, while the occurrence and progression of AD were gradually promoted in certain brain regions and presented certain distribution characteristics of brain regions. So immunohistochemistry was used to observe the distribution of PT231 in hippocampus after notoginsenoside Rg1 intervention. Through the immunohistochemical staining of PT231 in the left hippocampal region for the AD rat model group, normal group and notoginsenoside Rg1 middle dose group (determined based on the behavioral science and Western blot test results), it was found that the PT231 positive reactant was brown-yellow, and cluster-like positive cells were found in the DG, CA1 and CA3 regions of the hippocampus, with deeply stained round or oval cell bodies. The staining of membrane, cytoplasm, nucleus and dendrite and axon were obvious. In the DG region, the granulosa cell layer, the stratum corneum and the molecular layer were also obviously stained, and the cell body staining was more remarkable. In model group, the number of positive cells in DG region, CA1 region and CA3 region of hippocampus increased, the cell body increased and the staining depth increased. In DG region, the number of positive cells in granulosa cell layer, stratum corneum and molecular cell layer increased obviously, and the color of cell membrane was deeply stained. The distribution of middle dose notoginsenoside Rg1 group in DG region, CA1 region and CA3 region of hippocampus was between normal and model group. Compared with the model group, the number of positive cells decreased and the staining became lighter (Fig.3).

Fig.3 Effect of notoginsenoside Rg1 on the immunohistochemical staining of PT231 in hippocampus of AD model rats induced by Aβ25-35 (bar= 50 μm)

4 Discussions

AD is the main type of Alzheimer’s disease, which is the fourth cause of death in the elderly after cancer, heart disease and cerebrovascular disease. Senescence was the main cause of the disease. Senile plaques formed by Aβ deposition outside neurons and neurofibrillary tangles formed by the accumulation of overphosphorylated tau protein in neurons were the two major pathological manifestations. Although it is believed that the pathogenesis is mainly focused on Aβ hypothesis, tau protein hypothesis and chronic inflammation, neuron death hypothesis, the exact pathogenesis is not clear. At present, many drugs based on neuroprotection and Aβ secretion and deposition pathway have failed because of large side effects or no obvious effect[5]. The clinical study of tau protein as an active immunized vaccine is still in phase I clinical trial, and searching for specific preparations to inhibit tau protein hyperphosphorylation provides a good idea for clinical treatment of AD. The tau protein also mediated the toxicity of Aβ[6-7]. One of the proofs was that after Aβ42 immunotherapy in three AD patients, Aβ was removed from the brain, but the pathological changes and clinical symptoms of neurofibrillary tangles were still progressive[8]. This suggests that the abnormal tau protein is the main cause of cognitive impairment in the pathogenesis of AD, and some studies have shown that the degree of tau protein hyperphosphorylation in the cerebral cortex is positively correlated with the degree of cognitive impairment in dementia patients[9]. The present studies suggest that Panax notoginseng saponins have the lipid metabolism-regulating, anti-inflammatory, plaque-stabilizing and anti-oxidation effects, and thus have the effects of anti-atherosclerosis and protecting the heart. Notoginsenoside Rg1 can regulate the ratio of Bcl-2 to Bax in the brain tissue of rats with cerebral ischemia injury, and can promote the recovery of nerve function[10-12]. The present study suggests that notoginsenoside Rg1 can improve the spatial learning and memory ability of AD model rats and reduce the phosphorylation level of tau protein threonine 231 (PT231) in the hippocampus. Further morphological results suggest that notoginsenoside Rg1 can reduce the staining of PT231 in the DG, CA1 and CA3 regions of the hippocampus. Hippocampus is the main organ responsible for learning and memory, and memory is divided into transient memory, short-term memory and long-term memory, lasting 0.25-5 s, 5 s-2 min, many years and even lifetime. Instantaneous memory and short-term memory occur in the reticular formation of the brain stem, while long-term memory occurs in the temporal lobe and hippocampus. Therefore, it was speculated that the role of notoginsenoside Rg1 in improving the learning and memory ability might be related to the role of notoginsenoside Rg1 in lowering the levels of PT231 in the DG, CA1 and CA3 regions of the hippocampus of the rats.

At present, the research on the prevention and treatment of Alzheimer’s disease with TCM is still in the preliminary exploration stage, especially the terms of mechanism. The present study suggested that notoginsenoside Rg1 could reduce the levels of phosphorylated tau protein in the DG, CA1 and CA3 regions of the hippocampus of AD model rats and then enhance the spatial learning and memory ability of AD model rats, which provided a powerful basis and opened up new ideas for the prevention and treatment of senile dementia with Chinese medicine.

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