The Effect And Mechanism Of Cistanche Total Glycosides On Learning And Memory in Vascular Dementia Rats

The aging of the population has search for safe and effective drugs for the prevention and treatment of Alzheimer's disease, a hot spot in drug research. In recent years, significant progress has been made in the treatment of Alzheimer's disease using the method of tonifying the kidney and marrow, as well as the heart and spleen. Cistanche deserticola was originally described in Shennong's Classic of Materia Medica and is known for its effects of tonifying kidney yang, benefiting blood essence, moistening the intestines, and relieving constipation. It is now widely used to treat senile dementia, impotence, habitual constipation, and other diseases [1,2]. Studies have shown that it has the effects of anti-dementia, anti-oxidation, anti-hypoxia, anti-radiation, and enhancing immune function, and its main components are phenyl ethanol glycosides [3-6].

Cistanche deserticola ma-Anti-alzheimer's disease

The total glycosides of Cistanche deserticola ma are extracted and refined from the dried fleshy stems of Cistanche deserticola (Schrenk) R. Wight, containing 90.7% of phenylethanoid glycosides, including 44.3% of the main component echinacoside. In this experiment, the effects of total glycosides of cistanche on learning and memory impairment in vascular dementia rats and thrombosis and platelet aggregation in normal rats were studied to provide evidence for its development as a new drug for promoting intelligence and preventing and treating Alzheimer's disease.

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1. Material

1.1 Animals:

SD rats, male, weighing 470~570g, were provided by Shanghai Xipuma-Bikai Experimental Animal Co., Ltd., with the certificate number HDQZ No. 152.

1.2 Drugs and reagents:

The total glycosides of Cistanche deserticola, Cistanche extract powder, contain 90.7% of the total glycosides of phenyl ethanol, 44.3% of the Echinacoside, and dark brown powder, which is provided by the Chemical Office of the Modern Research Center of Traditional Chinese Medicine of Peking University, with the batch number of 980601. Piracetam tablets, produced by Yixing Pharmaceutical Factory, batch number 980521. Aspirin enteric-coated tablets, produced by Nanjing No. 2 Pharmaceutical Factory, batch number 000402. Each medicine is prepared with distilled water. Heparin sodium, a product of Shanghai Chemical Reagent Co., Ltd. of China Pharmaceutical (Group), batch number F000128. Pentobarbital sodium, imported by China Pharmaceutical (Group) Shanghai Chemical Reagent Co., Ltd., is sub-packed, with batch number 971104. Adenosine disodium diacetate (ADP), a product of Dongfeng Biochemical Technology Co., Ltd., Shanghai Institute of Biochemistry, Chinese Academy of Sciences, with batch number 9305122, was prepared into a 1mg/mL stock solution before use and stored in a refrigerator. When used, it was diluted 3 times with phosphate buffer.

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1.3 Instrument:

MG-3 Y-type electric maze, produced by Zhangjiagang Teaching and Experimental Equipment Factory. SPA-4 multifunctional platelet aggregator, produced by Shanghai Keda Test Instrument Factory.

2. Method

2.1 Vascular dementia model induced by focal cerebral ischemia-reperfusion in rats

Preparation and detection

2.1.1 Grouping and administration:

The rats were randomly divided into 6 groups according to their body weight, including a sham operation group, model group, positive control group (piracetam 300mg/kg), and high, medium, and low doses (200,100,50mg/kg) of total glycosides of cistanche. Each group was given 0.5 mL/100 g ig. once a day. The sham operation group and model group were given equal volumes of distilled water for 30 days.

2.1.2 Model preparation [7]:

On the 16th day of administration, rats were anesthetized with chloral hydrate 360mg/kg IP. Cut the neck skin in the middle, separate the right neck muscle, free the common carotid artery and external carotid artery, and ligate and cut off the branches of the external carotid artery. Separate the internal carotid artery until the pterygopalatine process artery is exposed. Clamp the common carotid artery on this side with an artery clamp, cut the external carotid artery, insert a blunt carbon fishing line with a diameter of 0.28 mm from the external carotid artery of the rat into the internal carotid artery, and enter the intracranial until there is a slight resistance, and then all blood supply of the middle cerebral artery is blocked. After 40 minutes of ischemia, the fishing line was pulled out, the external carotid artery was ligated, the common carotid artery was opened, disinfected, and sutured, and im penicillin was used to prevent infection. In the sham operation group, only the external carotid artery was isolated and ligated, and the common carotid artery was not clipped.

2.1.3 Learning and memory test [8]:

On the 7th day after the operation, a Y-type electric maze was used for learning and memory training and testing. The experiment was carried out in a quiet and dark environment. The rats were placed in one arm (the fixed starting area), adapted to the light for 2 minutes, and then given an electric shock. A light signal is set at the left arm, which is a safe area, and the right arm is a dangerous area with current and no light. The electric shock intensity is subject to the ability of the rats to run after the electric shock, and the voltage is 50-60V. It is correct for rats to escape to a safe area after being shocked, and it is wrong to escape to a dangerous area. After each training, they stay in the safe area for 30 minutes to consolidate their memory. When training again, take a rest for 10 seconds under the light of the starting area, repeat 10 times, record the correct and wrong times, and calculate the correct response rate. Each training was carried out 1h after administration and was continuously measured for 4 days. Then take the left and right arms alternately as the safety zone for another 4 days, record the correct and wrong times, and calculate the correct reaction rate.

Correct reaction rate: number of correct reactions/training times

2.2 Rat arteriovenous bypass thrombosis and detection [8]:

The rats were randomly divided into 5 groups according to their body weight, namely, the model group, the positive control group (aspirin 100mg/kg), high, medium, and low doses of total glycosides of cistanche (200, 100, 50mg/kg) groups. Each group was given 0.5 mL/(100 g), and the model group was given the same volume of distilled water for 7 days. After the last administration, ip pentobarbital sodium 45mg/kg anesthesia, supine position fixation, right common carotid artery, and left external jugular vein separation. Weigh a 7cm long No. 4 surgical silk thread and put it into the middle of the 3-segment polyethylene tube, so that the length of the silk thread in contact with blood is 6cm, and fill the whole polyethylene tube with 50U/mL heparin physiological saline. After inserting the venous end into the left external jugular vein, accurately inject 50U/kg heparin saline from the arterial end for anticoagulation, and then insert the arterial end into the right common carotid artery. The arteriovenous bypass was opened 1 hour after the administration, and the blood flow was interrupted 15 minutes after the opening. The silk thread was quickly taken out and placed on the weighing paper. The wet mass was weighed with an electronic analytical balance, and the thrombus inhibition rate was calculated.

Thrombosis inhibition rate=(thrombus mass in model group - thrombus mass in administration group)/thrombus mass in model group × 100%

2.3 Detection of rat platelet aggregation in vitro [8]:

The rats were randomly divided into 5 groups according to their body weight, which was the normal control group, the positive control group (aspirin 100mg/kg), and the total glycosides of cistanche administration group (the dose was the same as above). The administration method is the same as item 2.2. Fasting for 12 hours before the last administration, blood was taken from the abdominal aorta 1 hour after the last administration, anticoagulated with 3.8% sodium citrate (1:9), gently mixed, centrifuged at 1000 r/min for 3 minutes, and the upper layer of platelet-rich plasma (PRP) was sucked out. The remaining blood was centrifuged at 3000 r/min for 10 minutes, and the supernatant was anemic small plate plasma (PPP). Add 200 μL into the turbidimetric tube μ LPRP, adjust the zero point with PPP, preheat for 5min, and then add 50 μ ADP solution-induced platelet aggregation, measure platelet aggregation intensity, and calculate platelet aggregation inhibition rate.

Aggregation inhibition rate (maximum aggregation rate of the normal group - maximum aggregation rate of the administration group)/maximum aggregation rate of the normal group × 100%

2.4 Statistical processing:

The results were expressed in x ± s, and the data were statistically processed with the t-test of inter-group comparison.

3. Results

3.1 Effect on learning and memory function of vascular dementia model rats induced by focal cerebral ischemia-reperfusion:

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The results are shown in Table 1. The average correct response rate of each group increased day by day in the first 4 days of the test, and the average correct response rate of each group reached more than 90% on the fourth day. The average correct response rate of each dose group and positive group of total glycosides of cistanche was significantly higher than that of the model group in the first three days (P<0.05, 0.01), indicating that the administration of total glycosides of cistanche at 50, 100, and 200 mg/kg could significantly improve the correct response rate of maze training in rats with vascular dementia caused by focal cerebral ischemia-reperfusion, that is, total glycosides of cistanche had a significant protective effect on the learning and memory dysfunction caused by the model. There was no significant difference in the average correct response rate of each group on the 4th day, and the average correct response rate of each group decreased significantly after using the left and right arm alternately as the safety zone on the 5th day, but there was no significant difference between the groups. Then the average correct response rate of each group gradually increased, but there was no significant difference between the groups. The results showed that total glycosides of "desert ginseng", cistanche deserticola, could only restore the learning and memory function of rats with brain injury to a certain extent, and the change of learning style did not affect the acquired learning and memory ability.

Table 1 Effect of cistanchis glycosides on learning and memory function of vascular dementia rats induced by focal cerebral ischemia-reperfusion

*P<0.05 ** P<0.01 vs model group

3.2 Effect on rat arteriovenous bypass thrombosis:

The results are shown in Table 2. Compared with the model group, total glycosides of Cistanche deserticola 200 and 100 mg/kg could significantly inhibit the formation of arteriovenous bypass thrombosis in rats, but their effects were significantly weaker than that of aspirin 100 mg/kg. Total glycosides of Cistanche deserticola 50mg/kg did not affect the formation of arteriovenous bypass thrombosis in rats. It shows that the total glycosides of Cistanche deserticola can inhibit the formation of arteriovenous bypass thrombosis in whole animals.

Table 2 Effect of cistanchis glycosides on arteriovenous shunt thrombosis of rats(n=10)

*P<0.05 ** P<0.01vs model group

3.3 Effect on ADP-induced rat platelet aggregation in vitro:

The results are shown in Table 3. Compared with the normal group, the total glycosides of Cistanche deserticola 200, 100, and 50 mg/kg could significantly inhibit the platelet aggregation induced by ADP in vitro, and the inhibition rate of the 200 mg/kg group was the highest, reaching 59.48%.

Table 3 Effect of Cistanchis glycosides on platelet aggregation in vitro induced by ADP

*P<0.05 ** P<0.01 vs normal group

4. Discussion

Alzheimer's disease can generally be divided into four types, namely Alzheimer's disease (AD), vascular dementia (VD), mixed dementia (the former two coexist), and senile dementia caused by systemic diseases. Epidemiological survey shows that the incidence rate of AD in Western countries is higher than that of VD, but it is different in China. According to the general survey of 11 cities, the prevalence of VD is higher than that of AD [9]. VD is the general name of a dementia syndrome caused by brain tissue damage caused to cerebrovascular factors. Traditional Chinese medicine believes that this disease is mostly due to a deficiency of the essence and an excess of the essence. With the increase of age, the essence in the kidney of the body is gradually depleted, the marrow sea is gradually depleted, the function of the viscera is gradually weakened, and visceral failure will lead to the disorder of the distribution of water and blood in the body, the movement of qi and blood, and then the accumulation of pathological products such as phlegm and blood stasis in the body, the interference of phlegm and blood stasis will lead to the damage of the brain collateral, the lack of infiltration of qi and blood, the adverse rise and fall of the orifices and collaterals, the disorder of divine power. Therefore, the characteristic of this disease is that it is caused by deficiency and mixed with deficiency and excess. Kidney deficiency is the fundamental cause of the occurrence and development of Alzheimer's disease. The stagnation of phlegm and blood stasis, and the destruction of collaterals are the basic pathological links in the pathogenesis of this disease.

Cistanche deserticola is a traditional tonic and aphrodisiac, with the functions of tonifying the kidney, filling the essence, and moistening the intestines. The Classic of Materia Medica calls it the essential medicine for tonifying the blood essence and nourishing the kidneys. It has a good effect on the symptoms of fatigue, forgetfulness, hearing loss, and enuresis caused by kidney deficiency. Zhu Zhiming and others analyzed the drug composition of 46 compounds for the treatment of Alzheimer's disease and found that Cistanche deserticola is one of the most commonly used drugs [1].

Cistanche deserticola -Anti-Alzheimer's disease

In this experiment, on the 16th day of ig administration, rats were prepared with a VD model induced by focal cerebral ischemia-reperfusion, and on the 7th day of continuous administration, they were trained and tested for learning and memory. In the first three days of the test, the average correct response rate of the model group was significantly lower than that of the sham-operated group, indicating that cerebral ischemia-reperfusion injury caused learning and memory dysfunction of the model rats; The average correct response rate of rats in each dose group of cistanche glycosides was significantly higher than that in the model group, which was equivalent to the level of rats in the sham operation group, indicating that cistanche deserticola had a preventive and therapeutic effect on learning and memory impairment in VD rats, and cistanche glycosides were its effective anti-dementia component. The etiology and pathogenesis of VD are quite complex, and it is not very clear at present. Recent studies have shown that the risk factors of VD include those related to stroke and those unrelated to stroke, which are summarized as hypertension, heart disease, hyperlipidemia, diabetes, blood volume ratio, education level, age, gender, etc., while its pathogenesis is related to multiple infarcts, single infarcts in important parts, cerebral white matter ischemia, etc. [10,11]. In this experiment, the wet mass of the arteriovenous bypass thrombus in rats given total glycosides of Cistanche deserticola for 7 days was significantly lower than that in the model group; that is, total glycosides of Cistanche deserticola can significantly inhibit the thrombosis in the whole animal body. The results also showed that the total glycosides of Cistanche deserticola could significantly inhibit the platelet aggregation induced by ADP in vitro. Combining the two experimental results, it can be found that the mechanism of prevention and treatment of VD by total glycosides of cistanche may be related to the inhibition of platelet aggregation.

References:

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[2 ] Yu F. Research advances of cistanch e in clinical application [ J]. Inner Mon gholia Med J , 2003, 35 ( 6): 535-536.

[3 ] Liang M H, Sun P Y, Cao Y. Effect of gl ycosides of cistanche on lipid peroxidation in hemorrhagic shock/reperfusion in jury rabbits [ J]. J Ch in Microcir c, 2000, 4( 4): 230-231.

[4 ] Sh ang X Y, Wang X W, Wang X F, et al. Protective effect of gl ycosid es of cistanche on the immune function of 60 Co-ray irradiated mice [ J]. Ch in Trad it Herb Dru gs, 2001, 32( 2): 139-142.

[5 ] Wu L Y, Wang X W, Zao R M. The protective effect of the gl ycosides of Cistanch e on the cerebral hypoxia in mice [ J]. J Xinjiang Med Uni v , 2003, 26 ( 6): 561-562.

[6 ] Mut eli efu G, Liu M J, Lu J F. Effect of cistanoside compounds on oxidative stress and immunity [ J]. J Ch in Pharm Sci, 2001, 10( 3): 157-160.

[7 ] Longa E Z, Weinstein P R, Carls on S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[ J]. Stroke, 1989, 20( 1): 84-91.

[8 ] Xu S Y, Bi an R L, Chen X. Methodology in Pharm acolog ical Exp erimen t [M ]. 3rd ed. Beijing: People's Medical Publishing House, 2002.

[9 ] Wu F Q. Cerebrovascu lar demen tia [ J]. Adv Card iovasc Dis, 1993, 14( 2): 90-92.

[10 ] Skoon I.S. Status of risk factors for vascular dem en tia [ J]. Neu roep idem iolo gy, 1998, 17( 1): 2-6.

[11 ] Loeb C. M en tal Deterioration related to lacunar infarctions [ J]. Heart Dis Stroke, 1994, 23: 75-81.