Part 1 Effects Of Cistanche Deserticola Phenylethanoside On The Expression Of β-amyloid Protein in Hippocampus Of APP / PS1 Transgenic Mice

Part 1 The effective ingredients of Cistanche: the therapeutic effect of total phenethyl alcohol glycosides and verbascosides on Alzheimer's disease

For more information: Ali.ma@wecistanche.com

JU Bo-wei1 ，YANG Jian-hua2 ，HU Jun-ping3*

1 The Fifth affiliated Hospital of Xinjiang Medical University， Urumqi 830000， China;

2 The first affiliated Hospital of Xinjiang Medical University， Urumqi 830054， China;

3 School of Pharmacy， Xinjiang Medical University， Urumqi 830011， China

Abstract:

To study the effect of Cistanche deserticola phenylethanoid on the expression of the β-amyloid protein in the hippocampus of mice with Alzheimer's disease，60 6-month-old APP/ PS1 double transgenic AD mice were randomly divided into model groups， donepezil group ( 0． 65 mg/kg)， the high， middle， low dosages of total glycosides of Cistanche deserticola ( 250，125 62． 5 mg/kg) and acteoside( 125 mg/kg) were given drugs and distilled water for 3 months． At the age of 9 months， the Morris water maze and passive avoidance tests were used to determine the learning and memory impairments， the hippocampal lesions of mice were observed by HE staining， and the expressions of Aβ1-42 and Aβ1-40 proteins in the hippocampus of mice were de- tested by immunohistochemical method and Western-blot method． Cistanche deserticola phenylethanoid can significantly im- prove the learning and memory ability and the damage of hippocampal neurons in APP/ PS1 double transgenic mice． Compared with the model group， the number of Aβ1-42， Aβ1-40 positive cells in the hippocampus CA1 of the Cistanche deserticola group and the control group was significantly decreased，and the results of Western-blot showed that the expression of Aβ1-42， Aβ1-40 protein in the hippocampus of Cistanche deserticola in each dose group and the intervention group was significantly lower than that in model group． Cistanche deserticola glycoside can protect neurons and antagonize AD by down-regulating the expression of Aβ1-42， Aβ1-40 protein in the hippocampus brain of mice． In this study， phenylethanoid was identified as the main effective sub-stance for Cistanche deserticola to exert its anti-AD activity which provides theoretical support for its further development as a new drug against AD．

Keywords: Cistanche deserticola; phenylethanoid; Alzheimer's disease; APP/ PS1 double transgenic mice; β-amyloid protein

Click to Cistanche tubulosa powder for Alzheimer's disease

Alzheimer's disease is a central nervous system degenerative disease characterized by progressive memory loss and cognitive dysfunction. At present, the well-recognized β-amyloid protein (Aβ) cascade hypothesis believes that the normal metabolites Aβ1-42 and Aβ1-40 monomers in physiological conditions are not neurotoxic, but when they encounter APP exogenous mediators, they cause abnormalities. After deposition, the accumulated Aβ1-42 forms soluble Aβ oligomers (ADDLs), which prevent the monomers from producing protective activity and cause neuronal degeneration, synapse loss, and axon damage, and finally cause neuronal apoptosis in the brain to cause dementia, so Investigating the expression of its key targets Aβ1-42 and Aβ1-40 protein in the brain is of great significance for the research on the prevention and treatment of Cistanche phenylethanoid glycosides in AD. At the same time, studying the histopathological examination of the hippocampus of the characteristic lesions in the brain of AD animal models can truly reflect the improvement of the drugs on the lesions in the brain from the macroscopic view.

Traditional Chinese medicine in my country believes that Cistanche, a traditional Chinese medicine that nourishes the kidney and nourishes essence, has the effects of anti-aging, anti-aging, and improving memory. A large number of preliminary studies have confirmed that the total phenylethanoid glycosides of Cistanche and Acteoside, the representative monomer of phenethyl alcohol glycosides, exhibit obvious antagonistic effects on AD in the in vivo or in vitro models of AD constructed by traditional methods, and have certain prevention and treatment of neurodegeneration. The potential of sexual diseases. However, the application of the APP/PS1 double transgenic model of Cistanche Acteoside has not been used to conduct research on the prevention and treatment of AD from the perspective of β-amyloid.

In summary, this study is based on the classic hypothesis of AD pathogenesis—Aβ cascade reaction hypothesis, with the help of an ideal and reliable AD animal model APP / PS1 double transgenic mice as an in vivo research platform, using Morris water maze and HE staining method, Western-blot, immunohistochemistry, and other advanced technical methods, from the perspectives of animal behavior, histopathology, and molecular biology In order to find new targets for the prevention and treatment of AD, it will provide a reference for the study of the specific mechanism of action of Cistanche phenylethanoid glycosides in the prevention and treatment of AD.

1. Reagents and instruments

1.1 Reagent

Cistanche tubulosa phenylethanoid glycosides (self-made by the research group, the content of phenylethanoid glycosides is 87.6%); Acteoside; titanium dioxide; HE staining kit; neutral gum; SDS-PAGE gel preparation kit; BCA protein quantification kit; Aβ1-40; Aβ1-42; β-actin; Alkaline Phospase-labeled rabbit anti-goat IgG (H + L); 4 × protein loading buffer Contains β-mercaptoethanol; 10 × TBST; PVDF membrane; SDS; Tris; glycine; skimmed milk powder; BCIP /NBT Kit; secondary antibody; Tween 20.

1.2 Animals

60 6-month-old APP/PS1 double transgenic AD model mice

And 10 litter-negative mice, male and female, body weight 20 ± 10 g, the animals eat and drink freely during the experiment.

1.3 Apparatus

MT-100 Morris water maze; gel imager; RM2016 disease

Microtome; SIM-F124 ice maker; gel electrophoresis and transfer membrane equipment; full-wavelength microplate reader; CO2 constant temperature incubator; 4°C and -20°C refrigerator; 67120 ultrapure water instrument

; TP-114 electronic balance; low temperature refrigerated high-speed centrifuge; DVKW-D-2 digital display electric heating constant temperature water bath; ultrasonic cell disrupter; WD-9405A decolorizing shaker; adjustable constant voltage and constant current power supply; DM4000 fluorescent inverted microscope.

2. Method

2.1 Animal grouping and administration

According to SPSS18. 0 Random numbers generated by statistical software, 1156 Natural Products Research, and Development Vol. 31AD model mice were randomly divided into model group and donepezil group (daily infusion)

The gastric dose is 0. 65 mg/kg), Cistanche phenethyl alcohol total glycosides (PhGs) high, medium, and low dose groups (daily gavage doses are 250, 125, 62.5 mg/kg, respectively), Cistanche verbascoside intervention group (125 mg/kg) kg), 10 per group. At the age of 6 months, the mice were given intragastric gavage treatment according to groups, and the mice were intragastrically administered once a day for 3 consecutive months. The normal group consisted of non-transgenic mice in the same litter. The model group and the normal group were given an equal volume of distilled water by gavage.

2.2 Morris water maze experiment

The classic Morris water maze was used to test the spatial learning and memory abilities of mice. On the first and second days, train the mice to be familiar with the position of the platform and practice finding the platform. On the 3rd to 5th day, the formal test will be conducted to observe the situation of the mice in each group looking for the platform after training. On the 6th day, the platform will be removed and the mice will be observed.

Swimming track. During the experiment, the camera will simultaneously record the time spent by the mouse looking for the platform (ie escape latency) and the distance of the swimming track. If the mouse does not find a platform within the set time range (generally set to 60 s or 120 s, this experiment uses 60 s), then the escape latency of the mouse is recorded as the set value, and the staff will stay on the platform for a few seconds (10 s is used in this experiment) to help it deepen the memory of the platform. After the experiment, record the swimming time of the mice from entering the water to finding the platform. On the last day of the experiment, the platform was removed, and the content to be recorded during the set time mainly included the number of times the mouse crossed the platform, the stay time of the target quadrant, etc., and the search strategy of each experimental animal were analyzed.

2.3 Brain tissue extraction and paraffin embedding

After the administration, the mice were weighed, and the mice were anesthetized with 10% chloral hydrate. Place the anesthetized mouse on the operating table to fix the limbs. After 4% paraformaldehyde perfusion and fixation, the animals were sacrificed, the cerebellum and other extra brain tissue were removed, and the whole cerebellum and the brain were removed along the end of the cerebral hemisphere.

The front half of the central crown. Fix in 4% paraformaldehyde for 24 hours, wash with ultrapure water for 10 minutes, and then put in 30% ethanol (1 h) → 40% ethanol (1 h) → 50% ethanol (1 h) → 75% ethanol (1 h) ) → 90% ethanol (1 h) → 95% ethanol (1 h) → anhydrous ethanol I (1 h) → anhydrous ethanol II (1 h) → xylene I (15 min) → xylene II (15 min ). After being immersed in paraffin for 2 hours, the tissues were embedded in continuous coronal sections in the CA1 area of the hippocampus of the mouse brain. After drying in an oven at 60 ℃ for 48 h, store at room temperature.

2.4 HE staining

Bake the slices selected in each group in a 60 °C oven for 40 minutes, and then put them in xylene I (5 min) → xylene II (10 min) → absolute ethanol (5 s) → 95% ethanol I (5 s) → 95% ethanol II (5 s) → 80% ethanol (5 s) → distilled water

Second, use the HE staining kit to stain and mount the slides with neutral gum.

2.5 Immunohistochemical staining

The prepared sections were subjected to immunohistochemical staining in accordance with the standard steps of dewaxing, antigen retrieval, blocking endogenous catalase, serum blocking, primary antibody incubation, secondary antibody incubation, DAB color development, dehydration, and mounting. Choose 6 suitable slices for each group, observe the neuron fineness under the electron microscope

Cell morphology, coloration, and count the number of hippocampal positive cells using Image J software.

2.6 Western blot analysis of protein expression

Aspirate the tissue supernatant to determine the protein content extracted from the hippocampus of the brain tissue according to the BCA protein quantification method. Mix it with 4× protein loading buffer at a ratio of 4:1, heat it at 95 °C for 10 min, cool it naturally, and store it in a refrigerator at -20 °C. Prepare the SDS-PAGE gel, add the electrophoresis solution and then add the normal group, the drug intervention group, and other histones in sequence, and add the protein Marker to the gel holes on both sides. Stop electrophoresis in the ice bath to the bottom of the separation gel. After the transfer membrane liquid is balanced, cut out the PVDF membrane and filter paper according to the size of the glue. In the transfer clamp, follow the steps of black fiber mat → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → black fiber mat to assemble. After removing the bubbles, transfer the membrane at a constant pressure of 80 V. After the film transfer is completed, the skim milk is sealed for 2 h. The diluted antibody is added to the corresponding membrane and incubated night. After incubating the secondary antibody for 2 h, add BCIP/NBT color developing solution until the color develops. BIO-RAD gel imager takes pictures. Use Image J software to quantitatively analyze the target protein band, and use IOD to indicate the relative strength of the protein band. The result is expressed as the ratio of the integrated optical density value of the target protein and the control protein β-actin. The result is expressed as (target protein ID/internal reference ID) × 100%.

2.7 Statistical analysis of data

Use SPSS18. 0 Perform statistical analysis of the data. The data is represented by (x± s ). The data comparison between groups uses the One-Way ANOVA test and analysis, and the multiple comparisons between data use the LSD-t-test (least significant difference, LSD). ), with P <0. 05 or P <0. 01 means that the difference is statistically significant.

3 Experimental results

3.1 Morris water maze detection results

As the training time continues to increase, the time for each group of mice to reach the platform (escape latency) is significantly shortened. The first and second days are the training time. On the third day, the escape latency of the model group was significantly longer than that of the normal control group (P <0.01); the fourth and fifth days were compared with the normal control. Compared with the model group, the escape latency of the mice in the donepezil group was significantly shorter than that of the model group (P <0.05); compared with the model group, the total glycosides of cistanche phenethyl alcohol In each dose group and Acteosides group, as the dose increased, the escape latency of mice was significantly shortened, with significant differences (P <0.05) (see Table 1).

On the 6th day of the water maze experiment, the platform was removed for a space exploration experiment, and the number of times each group of mice crossed the platform position within 60 s was recorded. It can be seen from the results that the number of times of crossing the platform in the model group within the set time was significantly reduced compared with the normal group (P <0.01); compared with the model group, the cistanche phenethyl alcohol total glucoside groups and mullein The number of times of crossing the platform in the glycoside group was significantly increased (P <0.05), with significant differences (see Table 2). The above research results all suggest that Cistanche phenylethanoid glycosides can significantly improve the learning and memory ability of APP/PS1 double transgenic AD mice, and it has a certain dose-dependence.

3.2 Observation results of pathological tissue staining

The results show that the neurons in the CA1 area of the hippocampus in the normal group are arranged neatly and tightly, distributed more evenly, with clear layers, and the vertebral body cell nucleus is larger and the shape is approximately round. However, the CA1 area of the hippocampus of the model group mice was damaged, resulting in obvious changes in neuronal cells and morphology, the arrangement was loose and chaotic, there was no obvious hierarchy, the connections between the cells were loose, and some nuclei appeared pyknosis and apoptosis, making the staining deeper. The hippocampal CA1 cells of the different doses of Cistanche phenethanoloside intervention group, donepezil group, and verbascoside intervention group all improved to varying degrees. The pyknotic and deeply stained neuronal cells were significantly reduced compared with the model group, but larger ones can still be seen in Intercellular space (see Figure 1).

3.3 Results of immunohistochemistry experiment

After treatment with Cistanche phenethyl alcohol glycosides, a nine-month-old APP/PS1 double transgenic mouse brain tissue section was taken, and the hippocampal CA1 area was subjected to Aβ1-40 immunohistochemical staining. The results showed that the positive cells were mostly circular in the visual field Most of the cell membranes are brownish-yellow. Compared with the normal group, the number of Aβ1-40 positive cells in the hippocampus CA1 area of the model group increased significantly (P <0.01); compared with the model group, the total phenethyl alcohol glycosides of Cistanche tubulosa was high. The number of positive cells in the medium-dose group and the Acteoside glycoside group was significantly reduced (P <0.01), the donepezil group, and the Cistanche phenylethanoid glycosides low dose.

The number of Aβ1-40 positive cells in the dose group was significantly reduced (P<0.05), with significant differences (see Table 3, Figure 2).

After the intervention of Cistanche phenylethanoid glycosides, the brain tissues of 9-month-old APP/PS1 double transgenic mice were taken, and the hippocampal CA1 area was stained with Aβ1-42 immunohistochemical staining. The results showed: Compared with the model group, Cistanche phenylethanoid glycosides The number of positive cells in each dose group of glycosides and Acteoside glycoside group was significantly reduced, with a significant difference (P <0.01)

3.4 Western-blot test results

The Western-blot method was used to detect the expression of Aβ1-40 and Aβ1-42 protein in the hippocampus of mice. The results showed that the protein expression of Aβ1-40 and Aβ1-42 in the hippocampus of the model group was significantly up-regulated compared with the normal group, and there was a significant difference (P < 0. 05). The protein expressions of hippocampus Aβ1-40 and Aβ1-42 in mice in the intervention group of Cistanche phenylethanoid glycoside and verbascoside intervention group were significantly down-regulated compared with mice in the model group (P <0.01), and there was a significant difference (P <0.01). 01), see Figure 4.