Application of cistanche phenylethanoid glycoside in preparation of neuroprotective medicine for treating radiation long-term injury

Abstract

The invention discloses application of cistanche phenylethanoid glycoside in preparation of a neuroprotective medicine for treating radiation long-term injury, and belongs to the technical field of radiation medicine. In order to solve the problem that a method for effectively treating long-term nerve injury caused by ionizing radiation is lacked in the prior art, the invention provides application of cistanche phenylethanoid glycoside in preparation of a nerve protection medicine for treating long-term radiation injury. The cistanche phenylethanoid glycoside provided by the invention has the effects of remarkably improving physiological conditions of body weight, autonomous exploration ability, activity, learning and memory ability reduction and hair whitening of mice after ionizing radiation; the effects of improving mouse hippocampus tissue structure nucleus shrinkage, neuronal swelling and apoptotic body increase morphological change are achieved; the functions of reducing the content of inflammatory factors, improving the expression quantity of BDNF protein, reducing the phosphorylation degree of Tau protein and reducing the content of ROS and MDA are achieved; the functions of reducing the expression quantity of apoptosis-related proteins and improving the expression quantity of anti-apoptotic proteins are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of radiation medicine, and specifically relates to the application of Cistanche deserticola phenylethanoid glycoside in the preparation of a neuroprotective drug for treating long-term radiation damage. Background Art

[0002] At present, there are many cases of late damage after radiotherapy and chemotherapy in clinical practice. The more common late damages include: radiation-related dental caries damage, head drop syndrome caused by radiation involving weakness of neck extensor muscles, radiation-type brain-type late reactions, etc. The above-mentioned late damages have brought many new challenges to the quality of life of cancer patients. The specific observation time, radiation dose, cause of disease, and disease target of such late damage caused by ionizing radiation are still unclear. Therefore, the discovery of potential therapeutic drugs for studying the pathogenesis and disease targets of late damage is of great significance for clinical anti-radiation therapy.

[0003] Cistanches Herba is the dried fleshy stem with scaly leaves of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk)Weight of the Orobanchaceae family. It is warm in nature, sweet and salty in taste, and enters the kidney and large intestine meridians. It has the functions of tonifying kidney yang, benefiting essence and blood, and moistening the intestines and relieving constipation. It is used for kidney yang deficiency, essence and blood deficiency, impotence and infertility, soreness of waist and knees, weakness of tendons and bones, and constipation caused by dry intestines. Cistanches Herba was first recorded in Shennong's Herbal Classic and listed as a top grade. It is a traditional and precious Chinese medicinal material in my country and is known as the "desert ginseng".

[0004] The effective ingredients of Cistanche include flavonoids, lignin, alkaloids, etc. Analysis of its main components shows that they are mainly phenylethanoid glycosides, lignin and its glycosides, polysaccharides and related volatile components; among them, relevant studies on Cistanche phenylethanoid glycosides in recent years have shown that Cistanche phenylethanoid glycosides have good brain-enhancing and intelligence-enhancing effects, inhibit the apoptosis of brain cells, improve antioxidant capacity and significantly improve the movement disorder symptoms of Parkinson's model mice.

[0005] At present, there are no reports on the relevant research on the effect of Cistanche phenylethanoid glycosides on long-term damage caused by ionizing radiation, especially on neuroprotection. Those skilled in the art are eager to develop a method for effectively treating long-term nerve damage caused by ionizing radiation with Cistanche phenylethanoid glycosides as the main ingredient. Summary of the invention

[0006] The present invention solves the problem that the prior art lacks an effective method for treating long-term nerve damage caused by ionizing radiation, and provides the use of Cistanche deserticola phenylethanoid glycoside in the preparation of a neuroprotective drug for treating long-term radiation damage.

[0007] One of the purposes of the present invention is to provide the use of Cistanche deserticola phenylethanoid glycosides in the preparation of a neuroprotective drug for treating late radiation damage, wherein the late damage is caused by ionizing radiation.

[0008] The second object of the present invention is to provide a neuroprotective drug for treating late radiation damage, wherein the drug comprises Cistanche deserticola phenylethanoid glycoside and pharmaceutically acceptable excipients.

[0009] In a preferred embodiment of the present invention, the late damage is caused by ionizing radiation.

[0010] In a preferred embodiment of the present invention, the effective dosage of the Cistanche deserticola phenylethanoid glycosides is 100-200 mg / kg / day.

[0011] In a preferred embodiment of the present invention, the drug dosage form is any one of a gavage preparation, a tablet, a capsule or a liquid preparation.

[0012] Beneficial effects of the present invention: The present invention provides the use of Cistanche deserticola phenylethanoid glycoside in the preparation of a bone protection drug for treating long-term radiation damage, wherein the effective dose of the Cistanche deserticola phenylethanoid glycoside is 100-200 mg / kg / day.

[0013] (1) The Cistanche deserticola phenylethanoid glycosides provided by the present invention have a significant improvement effect on the physiological conditions of weight loss and hair whitening of mice after ionizing radiation, as well as the reduction of autonomous exploration ability and activity of mice after ionizing radiation.

[0014] (2) The Cistanche deserticola phenylethanoid glycosides provided by the present invention have a significant improvement effect on the reduction of mice's ability to explore new objects, and the reduction of their learning and memory abilities caused by ionizing radiation.

[0015] (3) The phenylethanoid glycosides of Cistanche deserticola provided by the present invention have a significant improvement effect on the morphological changes of nuclear shrinkage, neuronal swelling, and increased apoptotic bodies in the mouse hippocampal tissue structure caused by ionizing radiation.

[0016] (4) In mice treated with the Cistanche phenylethanoid glycosides provided by the present invention, the serum levels of IL-6 and TNF-α inflammatory factors were significantly reduced and dose-dependent, the relative expression of BDNF protein was increased, and the phosphorylation degree of Tau protein was reduced, and the differences were significant. In mice treated with the Cistanche phenylethanoid glycosides provided by the present invention, the ROS and MDA contents were significantly reduced. In mice treated with the Cistanche phenylethanoid glycosides provided by the present invention, the relative expression of apoptosis-related proteins p53, caspase3 and bax proteins was reduced, and the relative expression of anti-apoptosis protein bcl2 was significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of Cistanche deserticola phenylethanoid glycosides on the body weight of mice after ionizing radiation in Example 3;

[0018] Figure 2 This is a diagram showing the effect of Cistanche deserticola phenylethanoid glycosides on the physique of mice after ionizing radiation in Example 3;

[0019] Figure 3 Statistical graphs of the open field experiment results of mice in Example 3; A is a statistical graph of the activity time in the central area; B is a statistical graph of the number of times the mice enter the central area; C is a statistical graph of the average speed of the open field movement;

[0020] Figure 4 This is the movement trajectory diagram of the mouse open field test in Example 3;

[0021] Figure 5 This is the motion trajectory diagram of the mouse novel object recognition experiment in Example 4;

[0022] Figure 6 This is a recognition index graph of the new object recognition experiment of mice after ionizing radiation by phenylethanoid glycosides of Cistanche deserticola in Example 4; the ordinate Recognition index is the recognition index;

[0023] Figure 7 This is the pathological picture of HE staining of mouse brain tissue in Example 5; wherein Hippocampus is the hippocampus tissue of the brain;

[0024] Figure 8 This is the Nissl staining pathological picture of mouse brain tissue in Example 5;

[0025] Fig. 9 A is a Nissl staining image of the cortical area of ​​the mouse brain tissue in Example 5; A is a pathological image; B is a statistical image; wherein Cortex is the cortex;

[0026] Fig.10 A is a graph showing the FPKM statistical results in Example 6; A is a box plot showing the FPKM statistics of a gene; B is a stacked bar graph showing the distribution of gene expression levels;

[0027] Fig.11 This is a graph showing the statistical results of the correlation coefficient in Example 6;

[0028] Fig.12 A is a PCA analysis diagram of each sample group in Example 6; A is a PCA scatter plot of each sample group; B is a PCA correlation heat map of each sample;

[0029] Fig.13 The heat map of differentially expressed genes in Example 6; A is the heat map of differentially expressed genes between the irradiated model group and the blank control group; B is the heat map of differentially expressed genes between the Cistanche deserticola phenylethanoid glycosides administration group and the model group;

[0030] Fig.14 The volcano plot of differentially expressed genes in Example 6; A is the volcano plot of differentially expressed genes between the irradiated model group and the blank control group; B is the volcano plot of differentially expressed genes between the Cistanche deserticola phenylethanoid glycosides administration group and the model group;

[0031] Fig.15 It is a GO annotation classification statistical diagram of differentially expressed genes in Example 6; A is a GO annotation classification statistical diagram of differentially expressed genes between the irradiated model group and the blank control group; B is a GO annotation classification statistical diagram of differentially expressed genes between the Cistanche deserticola phenylethanoid glycosides administration group and the model group;

[0032] Fig.16 is a KEGG pathway enrichment scatter plot of differentially expressed genes in Example 6; A is a KEGG pathway enrichment scatter plot of differentially expressed genes in the irradiated model group versus the blank control group; B is a KEGG pathway enrichment scatter plot of differentially expressed genes in the Cistanche deserticola phenylethanoid glycosides administration group versus the model group;

[0033] Fig.17 is the differential gene expression Venn diagram in Example 6;

[0034] Fig.18 Figure 6 shows the results of GO enrichment and KEGG enrichment analysis of 66 differentially expressed genes in Example 6; A shows the results of TOP30 GO enrichment; B shows the results of TOP10 KEGG enrichment;

[0035] Fig.19 Figure 1 is a graph showing the effect of Cistanche deserticola phenylethanoid glycosides on the PI3K / AKT / GSK3β signaling pathway and the secretion of inflammatory factors in Example 7; A is a heat map showing changes in genes related to the PI3K / AKT / GSK3β signaling pathway; B is a statistical graph showing IL-6 inflammatory factors; C is a statistical graph showing TNF-α inflammatory factors;

[0036] Fig. 20 The figure is the effect of phenylethanoid glycosides of Cistanche deserticola on the expression of cognitive-related proteins in mouse hippocampal tissue in Example 7; A is the result of Western blot of BDNF protein; B is the statistical graph of BDNF protein expression; C is the result of Western blot of Tau protein; D is the statistical graph of Tau protein expression; the ordinate Relative expression level is the relative expression level;

[0037] Fig.21 : The effect of Cistanche deserticola phenylethanoid glycosides on oxidative stress secretion in mouse hippocampus tissue in Example 7; A is a ROS fluorescence intensity detection diagram; B is an MDA detection diagram; C is a GSH-px detection diagram;

[0038] Fig. 22: A is a graph showing the effect of Cistanche deserticola phenylethanoid glycosides on apoptosis-related proteins in mouse brain tissue cells in Example 7; A is a heat map showing changes in oxidative stress-related genes; B is a protein blot hybridization band map; and C is a statistical graph showing relative protein expression levels. DETAILED DESCRIPTION

[0039] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0040] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0041] Example 1: Preparation of Cistanche deserticola phenylethanoid glycoside aqueous solution

[0042] Drug: Cistanche deserticola phenylethanol glycoside powder is provided by Hualan Chemical, the product batch is 290Kgs, batch number XTY20211125, its purity is 85%, it is a fine brown powder with a special smell, and it is stored in a refrigerator at 4℃;

[0043] Preparation of Cistanche phenylethanoid glycoside aqueous solution: Calculate the average body weight of each group of mice and the total weekly dosage. Prepare the Cistanche phenylethanoid glycoside aqueous solution immediately before administration, taking mice with an average body weight of 25g as an example;

[0044] Weigh 20 mg of Cistanche deserticola phenylethanoid glycoside powder on an electronic balance and transfer it to a 15 mL centrifuge tube. Use a pipette to add 10 mL of deionized water, shake thoroughly, and mark it as a high dose.

[0045] Weigh 15 mg of Cistanche deserticola phenylethanoid glycoside powder on an electronic balance, transfer to a 15 mL centrifuge tube, add 10 mL of deionized water using a pipette, shake thoroughly, and mark as a medium dose;

[0046] Weigh 10 mg of Cistanche deserticola phenylethanoid glycoside powder on an electronic balance, transfer to a 15 mL centrifuge tube, add 10 mL of deionized water using a pipette, shake thoroughly, and mark as a low dose;

[0047] Store the prepared Cistanche phenylethanoid glycoside aqueous solution in a 4°C refrigerator, return to room temperature before each use, and shake well before administration.

[0048] Example 2: Preparation of a model of long-term damage caused by ionizing radiation

[0049] In this example, 50 SPF-grade C57BL / 6J male mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (License No.: SCK (Beijing) 2021-006), 7 weeks old, weighing 20-22g. The above male mice were raised in the barrier breeding system of the Experimental Animal Center of the Military Medical Research Institute, 5 mice per cage, free food and water intake (standard solid feed and sterilized tap water were provided by the animal center), and the mice were divided into 5 groups using a random number table method: a normal control group (CON), an irradiated model group (Mod), a model group + a low-dose group (L) (100 mg / kg) of Cistanche deserticola phenylethanol glycosides, a model group + a medium-dose group (M) (150 mg / kg) of Cistanche deserticola phenylethanol glycosides, and a model group + a high-dose group (H) (200 mg / kg) of Cistanche deserticola phenylethanol glycosides, 10 mice in each group.

[0050] The irradiation scheme of the cumulative 10 Gy dose group was selected for this administration (1.5 m from the irradiation source, 1.18 Gy / min irradiation dose rate, 4 min 23 s single irradiation time, 30 days between two irradiations). After the second radiation treatment, the mice were intervened with the drug (the aqueous solution of Cistanche phenylethanoid glycoside prepared in Example 1) for 30 consecutive days. The blank control group and the irradiation model group were gavaged with normal saline (purchased from Shijiazhuang Siyao).

[0051] After one week of adaptive feeding, the mice in the irradiated model group (Mod), model group + low-dose Cistanche phenylethanoid glycosides group (L), model group + medium-dose Cistanche phenylethanoid glycosides group (M) and model group + high-dose Cistanche phenylethanoid glycosides group (H) were fixed in a special irradiation box, 10 mice per group, and the special irradiation box was placed on a table. The distance between the box and the irradiation source was 1.5m, the irradiation dose rate was 1.18Gy / min, and the total irradiation dose was 5Gy. After irradiation, the mice were returned to the animal center barrier breeding system for continued breeding; after 30 days, they received whole-body irradiation again (1.5m away from the irradiation source, irradiation dose rate was 1.18Gy / min, and the total irradiation dose was 5Gy). The blank control group did not receive any special treatment.

[0052] Example 3: Effects of Cistanche deserticola phenylethanoid glycosides on mice

[0053] (1) Effect of Cistanche deserticola phenylethanoid glycosides on body weight of mice

[0054] In this example, the body weight of each group of mice to be tested was measured, and Graphpadprism 8.4.3 was used for statistical analysis.

[0055] The results are shown in Table 1. After the first radiation treatment and before the second irradiation, compared with the blank control group, the body weights of mice in the irradiated model group (Mod), model group + low-dose Cistanche phenylethanoid glycosides group (L), model group + medium-dose Cistanche phenylethanoid glycosides group (M) and model group + high-dose Cistanche phenylethanoid glycosides group (H) decreased, and the weight loss trend was the same among the irradiated groups.

[0056] The results are shown in Table 1. Drug administration began after the second radiation treatment. During the administration period, the body weight of mice in each drug administration group tended to increase. Among them, the body weight increase trend of mice in the high-dose group (H) was more obvious and had a significant difference (P < 0.05), and there was no significant difference in the body weight increase of mice in the low-dose group (L).

[0057] like Figure 1 As shown, compared with the blank control group (CON), the body weight of mice in the irradiated model group (Mod) and the low-dose group (L) was significantly reduced, while the body weight reduction trend of mice in the medium-dose group (M) and the high-dose group (H) was smaller, and there was no significant difference.

[0058] Table 1

[0059]

[0060]

[0061] Note: * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001; #P<0.05, ## indicates P<0.01, ### indicates P<0.001.

[0062] (2) Effects of Cistanche deserticola phenylethanoid glycosides on the physical appearance of mice

[0063] In this example, the physical appearance of each group of mice to be tested was observed, and the results were as follows: Figure 2 As shown in the figure, after the second radiation treatment, the mice were in a depressed state and had reduced activity, and the hair on their backs turned white and became sparse. After the second radiation treatment, the low-dose group (L), medium-dose group (M) and high-dose group (H) were given an aqueous solution of Cistanche deserticola by gavage. Compared with the irradiated group, the emaciation of the mice in the high-dose group (H) was improved and the hair whitening was alleviated, the emaciation of the mice in the medium-dose group (M) was improved but the hair whitening on their backs was not alleviated, and there was no significant change in the emaciation and hair whitening of the mice in the low-dose group (L).

[0064] (3) Effects of Cistanche deserticola phenylethanoid glycosides on the autonomous exploration ability and activity of mice

[0065] In order to explore the effects of phenylethanoid glycosides of Cistanche deserticola on the autonomous exploration ability and activity of mice after ionizing radiation, an open field test was performed on each group of mice to be tested in this example.

[0066] like Figure 3 As shown, compared with the blank control group (CON), the activity time of mice in the central area, the number of times entering the central area and the average speed of open field movement in the irradiated model group (Mod) were significantly reduced (P < 0.05); compared with the irradiated model group (Mod), the activity time of mice in the central area in the low-dose group (L), medium-dose group (M) and high-dose group (H) were increased, among which the average speed of open field movement in the medium-dose group (M) was significantly increased (P < 0.05).

[0067] like Figure 4 As shown in the figure, from the movement trajectory diagram of mice in the open field area, it can be seen that compared with the blank control group (CON), the movement trajectory of mice in the irradiated model group (Mod) was concentrated at the edge of the open field, and "slipping edge"-like depressive behavior appeared; compared with the irradiated model group (Mod), mice in the medium-dose group (M) and high-dose group (H) entered the central area more frequently, and the trajectory at the edge of the open field was reduced, while there was no significant change in the low-dose group (L) compared with the irradiated model group (Mod) mice.

[0068] In summary, the Cistanche deserticola phenylethanoid glycoside aqueous solution provided by the present invention has an improving effect on the physiological conditions of weight loss and hair whitening of mice after ionizing radiation, as well as the reduction of autonomous exploration ability and activity of mice after ionizing radiation.

[0069] Example 4: New object recognition experiment

[0070] The new object recognition experiment is mainly divided into three stages, namely the adaptation stage, the familiarization stage and the test stage. The entire experimental cycle is two days. Before the experiment officially begins, the new object recognition experimental box is cleaned and the equipment is debugged.

[0071] The first day was the adaptation and familiarization period. During the adaptation period, each mouse in each group (blank control group (CON), irradiation model group (Mod), low-dose group (L), medium-dose group (M) and high-dose group (H)) in Example 2 was placed in the center of a 50×50 cm open square box for free exploration activities. At this time, no objects were placed in the test box; after a certain delay interval, the mice were returned to the cage, and two identical objects were placed at both ends of the diagonal of the experimental box. The two identical objects in the box were explored and memorized. Each mouse explored freely for 10 minutes, and the mice were returned to the cage after the experiment.

[0072] The next day was the test period, in which one object was replaced with a new object of different color and shape. Each mouse in each group in Example 2 explored freely in the test box for 5 min, and the number of interactions and behavioral paths of the mouse with the new object were recorded. The experimental data of the new object recognition experiment were statistically analyzed.

[0073] The results are as follows Figure 5-6 As shown in the figure, compared with the blank control group (CON), the novel object recognition index of mice in the irradiated model group (Mod) was significantly reduced (P < 0.05), indicating that the irradiated mice had weaker exploration behavior towards novel objects and decreased learning and memory abilities; compared with the irradiated model group (Mod), the recognition index of mice in the low-dose group (L), medium-dose group (M) and high-dose group (H) was significantly increased, and the difference in the high-dose group (H) was the greatest (P < 0.05).

[0074] It can be seen that ionizing radiation leads to a decrease in the mice's ability to explore new objects and their learning and memory abilities. The intervention of the Cistanche phenylethanoid glycosides provided by the present invention on the irradiated mice has a significant improvement effect on the decrease in the mice's ability to explore new objects and their learning and memory abilities.

[0075] Example 5: Morphological observation of pathological sections of mouse brain tissue

[0076] As an important structure of the brain, the hippocampus mainly controls daily learning and memory functions. In order to explore the effects of Cistanche deserticola phenylethanoid glycosides on the neuronal cell morphology and apoptotic bodies in different regions of the mouse hippocampus tissue, HE staining and Nissl staining were performed on the hippocampal tissues of each group of mice to be tested in Example 2.

[0077] (1) Specimen acquisition: The night before the specimen collection, the mice in each group in Example 2 were deprived of water and food. On the second day, venous blood was obtained from the mice by removing the eyeballs. The skull was then cut open with scissors to remove the mouse brain. One part of the brain was immersed in 4% paraformaldehyde tissue fixative, and the hippocampal tissue of the other part was carefully separated with forceps and stored in a -80°C refrigerator.

[0078] (2) HE staining of mouse brain tissue

[0079] S1: Take out the fixed brain tissue of the mouse to be tested from the 4% paraformaldehyde tissue fixative, and soak the slices in xylene I, II, and III for 15 minutes each; take them out and soak them in anhydrous ethanol I and II for 5 minutes respectively, soak them in 95% alcohol for 5 minutes, and finally soak them in 85% alcohol for 5 minutes. After completion, wash them with tap water;

[0080] S2: After washing in S1, the brain tissue slices of the mouse to be tested are completely immersed in the hematoxylin staining solution for 1-2 minutes, and after staining, they are taken out, washed with tap water, differentiated with differentiation solution for 2 minutes, and cleaned after completion; the differentiated brain tissue slices of the mouse to be tested are placed in the blueing solution for blueing, and cleaned after completion; the cleaned brain tissue slices of the mouse to be tested are immersed in the eosin staining solution for staining for 2-3 minutes, and cleaned after completion;

[0081] S3: The brain tissue slices of the mouse to be tested cleaned in S2 are sequentially placed in anhydrous ethanol I, anhydrous ethanol II, and anhydrous ethanol III and soaked for 5 minutes respectively. After completing the above soaking process, the brain tissue slices of the mouse to be tested are sequentially placed in xylene I and xylene II and soaked for 5 minutes respectively. The brain tissue slices of the mouse to be tested are observed until they are transparent. The brain tissue slices of the mouse to be tested are taken out and evenly sealed with neutral gum, and the brain tissue slices are observed under an electron microscope.

[0082] The results are as follows Figure 7 As shown, compared with the blank control group (CON), the CA3 and DG regions of the hippocampal structure of the irradiated model group (Mod) mice showed obvious nuclear shrinkage and loose arrangement of neurons; compared with the irradiated model group (Mod), the neurons in the medium-dose group (M) and high-dose group (H) mice had an improved effect on the nuclear shrinkage caused by ionizing radiation, and the number of shrunken neurons decreased, while there was no obvious change in the hippocampal structure of the low-dose group (L) mice.

[0083] (3) Nissl staining of mouse brain tissue

[0084] S1: Take out the fixed brain tissue of the mouse to be tested from the 4% paraformaldehyde tissue fixative, and soak the slices in xylene I, II, and III for 15 minutes each, take them out and soak them in anhydrous ethanol I and II for 5 minutes respectively, soak them in 95% alcohol for 5 minutes, and finally soak them in 85% alcohol for 5 minutes. After completion, wash them with tap water;

[0085] S2: Immerse the brain tissue slices of the mouse to be tested after washing in S1 in the dye solution for 5 minutes and then wash them with tap water. After washing, use 1% glacial acetic acid to differentiate the brain tissue slices of the mouse to be tested. Observe the morphology of the brain tissue slices of the mouse to be tested under a microscope and terminate the differentiation with tap water in time. Wash the brain tissue slices of the mouse to be tested and then place them in a drying oven for drying.

[0086] S3: Place the mouse brain tissue slices dried in S2 into xylene I and xylene II and soak them for 5 minutes respectively. Observe the brain tissue slices until they are transparent. Take out the brain tissue slices and seal them evenly with neutral gum. Observe the brain tissue slices under an electron microscope.

[0087] The results are as follows Figure 8 As shown, compared with the blank control group (CON), the apoptotic bodies in the CA1 and DG areas of the hippocampal structure of the irradiated model group (Mod) mice increased significantly, and obvious neuronal cell apoptosis occurred at the edge; compared with the irradiated model group (Mod), the number of apoptotic bodies in the medium-dose group (M) mice decreased, and the neuronal cells were arranged more closely at the edge, while there was no significant change in the hippocampal structure of the low-dose group (L) and high-dose group (H) mice.

[0088] In addition, in the Nissl-stained sections, this example statistically analyzed the number of Nissl bodies in the cortical layer below the hippocampus of mouse brain tissue. Fig. 9 As shown in the figure, compared with the blank control group (CON), the positive area of ​​Nissl staining in the cortex of the brain tissue of mice in the irradiated model group (Mod) was significantly increased (P < 0.05); compared with the irradiated model group (Mod), the positive area of ​​Nissl staining in the cortex of the brain tissue of mice in the medium dose group (M), high dose group (H) and low dose group (L) was slightly decreased, but there was no significant difference.

[0089] In summary, the results of pathological morphological observation show that after ionizing radiation, the mouse hippocampus structure showed obvious morphological changes such as nuclear shrinkage, neuronal swelling, and increased apoptotic bodies. The Cistanche phenylethanoid glycosides provided by the present invention intervened in the irradiated mice and had an improving effect on the morphological changes of the mouse hippocampus tissue.

[0090] Example 6: Transcriptome sequencing of mouse hippocampal tissue

[0091] (1) RNA extraction from mouse hippocampus tissue

[0092] Take out the hippocampal tissue samples of each group of mice to be tested obtained in Example 5 frozen in a -80°C refrigerator, place them in a mortar pre-cooled with liquid nitrogen, and grind the tissue with a pestle, continuously adding liquid nitrogen until it is ground into powder; after grinding, use an electronic balance to take 50-100 mg of the hippocampal tissue of the mouse to be tested and put it into a 2 mL round-bottom centrifuge tube, and add 1 mL of Trizonl with a pipette; set the homogenizer power to 20 Hz, transfer it to a 1.5 mL centrifuge tube after fully homogenizing for 5 minutes, add 200 μL of chloroform after standing for 5 minutes, cover the tube tightly, mix well, and stand at room temperature for 10 minutes; put the above samples into a centrifuge pre-cooled at 4°C, set the centrifuge speed to 13000 rpm, and The sample was placed in a centrifuge and centrifuged for 15 minutes; the supernatant was transferred to another new 1.5 mL centrifuge tube using a pipette and marked with a marker pen; isopropanol equal to the volume of the supernatant was added to each centrifuge tube, the sample was placed in a -20°C refrigerator and allowed to stand for 30 minutes after being inverted and shaken; the sample was then placed in a 4°C precooled centrifuge and centrifuged at 13,000 rpm for 15 minutes, and the supernatant was discarded; 500 μL of 75% ethanol was added to each of the above centrifuge tubes to wash the precipitate, and the sample was placed at 4°C and centrifuged at 13,000 rpm for 5 minutes, the supernatant was discarded and the precipitate was retained and dried; the lower precipitate was completely dissolved in H2O, and the hippocampal tissue RNA of each group of mice to be tested was obtained and stored at -80°C.

[0093] (2) Transcriptome library preparation

[0094] The RNA of the hippocampal tissue of each group of mice tested that passed the test in (1) was sampled, and 3 μg of total RNA from each sample was used as the starting material to construct a transcriptome sequencing library. Ultra TM RNA Library Prep Kit for According to the operating instructions, different index tags were selected to build libraries; magnetic beads were added to the qualified total RNA samples to enrich eukaryotic mRNA, and then fragment reagents were added to break the long-chain mRNA into mRNA fragments, and the corresponding cDNA chains were synthesized using the mRNA fragments as templates; cDNA buffer, dNTPs, RNase H and DNA polymerase I were added to each sample to be tested, and the double-stranded cDNA was synthesized after mixing evenly; after the synthesis, the double-stranded cDNA was purified, the end repaired and other post-tests were performed, and finally cDNA fragments of appropriate length were selected for PCR amplification to obtain the cDNA library.

[0095] (3) Data quality control

[0096] The raw data (Raw Data) contains the sequence information of the sequencing sequence and its corresponding sequencing quality information. Since the raw data has shortcomings such as uneven bases, the next step is to filter the original sequence to filter out impurity data such as sequencing adapter sequences and low-quality bases to obtain high-quality sequences (Clean Data). The relevant charge data of the high-quality sequence is calculated to evaluate the data quality and the relevance of the sample.

[0097] (4) Expression estimation

[0098] FPKM (Fragments Per Kilobase Millon Mapped Reads) represents the level of gene expression. The number of reads for each gene on the high-quality sequence was counted, and the expression level of each gene was estimated using HTSeq. The gene expression level is: FPKM = 10 6 ×FNL / 10 3 .

[0099] (5) Differential expression analysis

[0100] Differentially expressed genes (DEGs) are the key to transcriptome analysis and are also the key genes for ionizing radiation-induced pathogenesis. In the screening of differentially expressed genes, P_value≤0.05 was set as differentially expressed genes. However, because the above screening conditions were too broad, under the above preliminary screening definition, genes with |log2Ratio|≥1 and q<0.05 were further selected as significantly differentially expressed genes, that is, up- and down-regulated genes that are key to ionizing radiation-induced pathogenesis. According to the above screening conditions, linear regression calculations were performed on differentially expressed genes to estimate the expression intensity of genes in each sample, and the probability value p-value of each gene expressed in the blank control group, single 5Gy irradiation group, and cumulative 10Gy irradiation group was calculated. The p-value was corrected to obtain the q-value for subsequent enrichment analysis.

[0101] (6) GO and KEGG differential expression enrichment analysis

[0102] For differentially expressed genes, GO (Gene Ontology) enrichment analysis was performed to calculate the number of genes contained in each biological process, cell composition, and molecular function. The hypergeometric test was applied to screen the GO entries with significant enrichment of differentially expressed genes by comparing with the entire biological gene background. The screening conditions were redefined, and q≤0.05 was used as the threshold to screen out the top 10 specific biological processes in biological processes, cell composition, and molecular functions. The GO functional significance enrichment analysis was comprehensively analyzed to further determine the main biological functions of ionizing radiation differentially expressed genes.

[0103] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database of gene enrichment pathways. With a complete set of biological gene backgrounds, KEGG can predict the role of protein interaction networks in various cell activities. The selected differentially expressed genes were used as research objects to conduct pathway enrichment in the KEGG database. The top 20 signal pathways with the number of differentially expressed gene enrichments were screened with a threshold of q≤0.05. The KEGG signal pathway enrichment analysis was combined to further determine the specific cell pathway in which the ionizing radiation differentially expressed genes acted.

[0104] Performance data:

[0105] (1) This example constructs three mouse hippocampal tissue transcriptome libraries based on the hippocampal tissue samples of each group of mice to be tested obtained in Example 5, namely, a blank control group (C1-3), an irradiated model group (MOD1-3) and a Cistanche phenylethanoid glycosides administration group (PGS1-3), and analyzes the transcriptome data. All of the above samples were sequenced using double-end transcription on the HiSeq X-ten platform. The data obtained from the sequencing of the midbrain tissue of the blank group, the model group and the Cistanche phenylethanoid glycosides administration group of mice were subjected to sequencing quality control, and a total of 107.25Gb of Clean Data was obtained. The Clean Data of each sample reached 11.0Gb, and the Q30 base percentage was 11.56% or above (as shown in Table 2), and the reads with a quality score ≥Q30 in each sample were selected to generate a complete FASTQ file.

[0106] Table 2

[0107]

[0108]

[0109] Since the sequencing depth and gene length in transcriptome sequencing will affect the results, FPKM was used to uniformly correct the sequencing depth and gene length in subsequent analysis. The FPKM of all genes in each group was counted. The results are shown in Fig.10 As shown in Figure A, the box plot of FPKM in the blank control group (C1-3), irradiation model group (MOD1-3) and Cistanche phenylethanoid glycosides administration group (PGS1-3) are represented from top to bottom as the maximum value, the third quartile, the median, the first quartile and the minimum value. It can be seen from the box plot that the gene expression levels in each group are similar, and the next step of statistics can be carried out. The distribution of gene expression levels is stacked as shown in Fig.10 As shown in B, it can be seen from the figure that the FPKM of most genes is between 1-10, above 10 and between 0-0.25.

[0110] (2) The correlation coefficient was calculated based on the FPKM of all genes in the blank control group (C1-3), irradiation model group (MOD1-3) and Cistanche phenylethanoid glycosides administration group (PGS1-3). Fig.11 shown.

[0111] The correlation coefficient represents whether the samples within the group have good repeatability. Principal component analysis (PCA) is used to evaluate the differences between groups and the repetition of samples within the group. PCA analysis is performed on the samples according to the FPKM value. The linear algebra calculation method is used to reduce the dimension of each group of gene variables and extract the principal components, so that similar samples can be clustered together.

[0112] like Fig.12 As shown in the figure, the correlation coefficients of the three samples in each of the blank control group (C1-3), irradiation model group (MOD1-3) and Cistanche phenylethanoid glycosides administration group (PGS1-3) were all above 0.95, indicating that the three samples had good repeatability and could be used for the next experimental analysis; the blank control group (C1-3) and the model group (MOD1-3) could be clearly separated, indicating that the genes in the hippocampus tissue of mice had significant changes after ionizing radiation; compared with the irradiation model group (MOD1-3), the Cistanche phenylethanoid glycosides administration group (PGS1-3) was closer to the blank control group (C1-3), indicating that the gene expression in the hippocampus tissue of mice after intervention with Cistanche phenylethanoid glycosides tended to be similar to that of normal mice.

[0113] (3) In this example, the DESeq2 software was used to standardize the number of genes in each sample of the blank control group (C1-3), model group (MOD1-3) and Cistanche phenylethanoid glycoside administration group (PGS1-3), and screening was performed based on the difference multiple and difference significance test results.

[0114] The results are as follows Fig.13 As shown, A is a heat map of the irradiated model group versus the blank control group, and B is a heat map of the Cistanche phenylethanoid glycoside-treated group versus the model group. It can be seen that both figures show that the up- and down-regulation results of the differentially expressed genes in each group are significant, and the next step of analysis can be carried out.

[0115] The DESeq2 software (with log2FC≥1 and FDR<0.05 as screening criteria) was used to perform differential expression analysis on the three groups of data. Fig.14 As shown, A is the volcano map of differentially expressed genes between the irradiated model group and the blank control group, and B is the volcano map of differentially expressed genes between the Cistanche deserticola phenylethanoid glycoside-treated group and the model group. The volcano map can more intuitively show the up- and down-regulated genes, where red represents up-regulated differentially expressed genes and blue represents down-regulated differentially expressed genes.

[0116] It can be seen that there were 284 differentially expressed genes between the irradiated model group and the blank control group, of which 187 genes were upregulated and 97 genes were downregulated; there were 545 differentially expressed genes between the Cistanche deserticola phenylethanoid glycoside-treated group and the irradiated model group, of which 338 genes were upregulated and 207 genes were downregulated.

[0117] (4) GO enrichment analysis was performed on the differentially expressed genes obtained from the above comparison. The GO enrichment structure showed that a total of 284 differentially expressed genes (DGEs) in the irradiated model group and the blank control group were classified into the three major branches of GO, namely biological process (BP), molecular function (MF) and cellular component (CC). The statistical results of this study mainly focused on the enrichment analysis of the TOP10 differentially expressed genes in the three major branches (screening the GO entries corresponding to PopHits ≥ 3 in the three categories, and sorting the 10 entries from large to small according to the -log10p-value corresponding to each entry).

[0118] The results are as follows Fig.15 As shown in A, the statistical results of differentially expressed genes between the irradiated model group and the blank control group showed that the more enriched genes in BP were immune response (16 genes), negative regulation of peptidase activity (10 genes), and inflammatory response (14 genes); the more enriched genes in CC were extracellular space (60 genes), extracellular region (54 genes), and external side of plasma membrane (19 genes); the more enriched genes in MF were G protein-coupled receptor activity (15 genes) and G protein-coupled receptor binding (16 genes).

[0119] The results are as follows Fig.15As shown in B, the statistical results of the comparison between the Cistanche deserticola phenylethanoid glycosides group and the model group showed that the genes with higher enrichment in BP were neuron differentiation (15 genes), neuropeptide signaling pathway (8 genes), nervous system biological process (10 genes) and cell differentiation (23 genes); the genes with higher enrichment in CC were dynein complex (6 genes), extracellular space (46 genes) and inner dynein arm (5 genes); the genes with higher enrichment in MF were nucleoside diphosphate kinase activity (5 genes), sequence-specific double-stranded DNA binding (17 genes) and G protein-coupled receptor activity (15 genes).

[0120] In summary, the differentially expressed genes in the Cistanche phenylethanoid glycosides-administered group compared with the irradiated model group were mainly related to the positive regulation of neuronal apoptosis in terms of biological processes; they were mainly related to the extracellular space and extracellular region in terms of cell composition; and they were closely related to metal ion binding and G protein-coupled receptors in terms of molecular functions.

[0121] (5) KEGG is a major public database related to signal pathways. The KEGG database was used to analyze the signal pathways of differentially expressed protein-coding genes, and the hypergeometric distribution test method was used to calculate the significance of differentially expressed gene enrichment in each signal pathway. Comparing the differentially expressed genes between the Cistanche phenylethanoid glycosides group and the irradiated model group through KEGG analysis will help us better understand the protein interactions between the two groups and the changes in the signal pathways through which the neuroprotective effect is demonstrated after the intervention of Cistanche phenylethanoid glycosides.

[0122] The results are as follows Fig.16As shown in A, compared with the blank control group, the differentially expressed genes in the irradiated model group mainly involved 36 metabolic pathways, among which the most abundant differentially expressed genes were mainly enriched in the neuroactive ligand-receptor interaction pathway (11 genes), Toll-like receptor signaling pathway (5 genes), Alzheimer's disease (4 genes) and PI3K-Akt signaling pathway (4 genes).

[0123] The results are as follows Fig.16 As shown in B, compared with the irradiation model group, the enrichment results of KEGG signaling pathways of Cistanche deserticola phenylethanoid glycosides were mainly concentrated in neuroactive ligand-receptor interaction (14 genes), multiple pathways of neurodegeneration (9 genes), PI3K-Akt signaling pathway (6 genes) and cholinergic synapse (6 genes).

[0124] In order to more intuitively see the differentially expressed genes directly affected by the drugs, this example draws a Venn diagram of the differentially expressed genes in the three groups. Fig.17 As shown, there are 66 common differentially expressed genes in the Cistanche phenylethanoid glycosides-treated group compared with the irradiated model group, and in the irradiated model group compared with the blank group. It is speculated that the above 66 differentially expressed genes may be the key targets of Cistanche phenylethanoid glycosides in the treatment of cognitive-related damage caused by ionizing radiation.

[0125] In this example, GO enrichment and KEGG pathway enrichment analysis were performed on the above 66 common differentially expressed genes. The results of GO enrichment analysis are as follows: Fig.18 As shown in A, in BP, it is mainly related to neuronal differentiation and neurobiological processes; in CC, it is mainly related to high-density lipoprotein; in MF, it is mainly related to G protein receptor connection. The top 10 results of KEGG pathway enrichment analysis are as follows Fig.18As shown in B, it is mainly concentrated on neuroactive ligand-receptor interaction (5 genes) and multiple pathways of neurodegeneration (3 genes), while the signaling pathway is mainly concentrated on the PI3K-Akt signaling pathway (4 genes).

[0126] In summary, ionizing radiation causes cognitive impairment in mice mainly through the neuroactive ligand-receptor interaction pathway and multiple diseases of neuropathy. The number of genes enriched in the PI3K-Akt signaling pathway is large, suggesting that ionizing radiation-induced neuronal cell pathology is a process co-regulated by multiple signaling pathways, and the protective effect of Cistanche deserticola phenylethanoid glycosides on neuronal damage caused by ionizing radiation is also mainly through the neuroactive ligand-receptor interaction pathway and multiple diseases of neuropathy.

[0127] Example 7: Detection of cognition-related proteins and oxidative stress indicators in mouse hippocampal tissue

[0128] (1) Total protein extraction from mouse hippocampus tissue

[0129] Use an electronic balance to weigh 20 mg of the mouse hippocampus tissue to be tested and put it into a 1.5 mL EP tube. Add 200 μL of 4°C pre-cooled lysis solution and three disposable homogenization beads to each tube. Put the centrifuge tube into a homogenizer and homogenize the mouse hippocampus tissue to be tested at 20 Hz for 5 minutes. After the homogenization, put the centrifuge tube into a 4°C pre-cooled centrifuge and centrifuge at 12,000 rpm for 10 minutes. Aspirate the protein supernatant to obtain the total protein of the mouse hippocampus tissue to be tested.

[0130] (2) BCA protein quantification

[0131] The working solution was prepared according to the BCA protein quantification instructions, and the sample was added to a 96-well plate. 22.5 μL of pre-cooled PBS was added to each well, followed by 2.5 μL of the total protein extract of the hippocampal tissue of each group of mice to be tested obtained in (1). Finally, 200 μL of the above-prepared working solution was added. After shaking, the plate was incubated in a 37°C constant temperature incubator for 30 min, and then the absorbance was detected using an ELISA reader at a wavelength of 560 nm.

[0132] (3) Protein denaturation

[0133] Add 80 μL of the total protein extract of the hippocampal tissue of each group of mice to be tested obtained in (1) to a 200 μL EP tube, then add 20 μL of 5× Loading Buffer, pipette evenly to prepare a 100 μL sample system, denature the prepared loading buffer in a 100°C metal bath for 15 min, cool to room temperature, and place on ice for later use.

[0134] (4) Electrophoresis-membrane transfer

[0135] Prepare the gel and electrophoresis solution in advance according to the instructions. Shake the total protein samples of the mouse hippocampus tissue obtained in (3) before loading. Centrifuge again. Load 20 μg of the protein sample obtained in (3) into each loading well according to the concentration of the BCA protein quantification result in (2). Load one well and two wells with 5 μL of marker on the left and right sides, respectively. Add electrophoresis solution that covers the electrophoresis tank. Start electrophoresis at a voltage of 80 V. When markers of different protein molecular weights are completely separated, adjust the voltage to 100 V and continue the electrophoresis. End the electrophoresis when the sample is tangent to the green edge of the lower layer of the electrophoresis tank.

[0136] Prepare the transfer solution in advance and place it in a 4°C refrigerator to cool. After the electrophoresis is completed, take out the gel and cut it into squares slightly larger than the required PVDF membrane, and place it in the cooled electrophoresis solution for use; put the prepared PVDF membrane in methanol to activate it for 1 minute, and then transfer it to the transfer solution for use; add about two-thirds of the transfer solution to the tray, place the transfer template flat on the tray, and place the following on the anode surface of the transfer template in sequence: sponge → filter paper, PVDF membrane, gel layer, filter paper → sponge, and drive away the residual bubbles at each step. After placing them, clamp the transfer plate, and then place the transfer plate in the transfer tank, with the black side corresponding to the negative electrode and the transparent side corresponding to the positive electrode. Finally, transfer the transfer solution to the transfer tank, turn on the power, 150mA, and transfer in an ice bath for 100 minutes.

[0137] (5) Blocking and incubation with primary antibody-secondary antibody-exposure and development

[0138] After the transfer, use a ballpoint pen to distinguish the front and back sides of the PVDF membrane and then place it in TBST and shake it slowly at room temperature for 5 minutes to wash away the excess transfer solution; use skim milk powder and TBST to prepare 5% skim milk in proportion, place the washed PVDF membrane in skim milk, and shake it slowly on a shaker at room temperature for 90 minutes.

[0139] Place the blocked PVDF membrane in TBST, shake it slowly on a shaker for 5 minutes, and wash off the excess skim milk; place the PVDF membrane in a ziplock bag, and add TBST to ensure that the membrane is moist during the band cutting process; cut the complete PVDF membrane above and below the target protein molecular weight according to the protein molecular weight indicated by the markers on the left and right sides. After each band is cut, use a ballpoint pen to mark the name of the target protein on the right side of the band and place it in a clean TBST buffer.

[0140] Clean and dry the antibody incubation box in advance and place the prepared antibody on ice. Place the strips and corresponding antibodies in the antibody box according to the protein name on the right side of the strips. After completion, shake gently to make the primary antibody evenly cover the strips and incubate in a 4°C refrigerator overnight; after incubation for 12 hours, recover the primary antibody and wash the strips, place the strips in a clean incubation box, add sufficient TBST buffer, shake quickly on a shaker for 15 minutes, and wash 3 times in total; after washing, add the secondary antibody corresponding to the primary antibody species and let it stand at room temperature for 1 hour.

[0141] Prepare chemiluminescent developer in a ratio of 1:1 between solution A and solution B and place on ice for later use. After the above-mentioned secondary antibody incubation is completed, wash the strips again with TBST buffer and repeat 3 times. After washing, use clean filter paper to absorb the TBST on the surface of the strips, place them in the developer for 15 seconds, take them out and put them into the developer for chemiluminescent development.

[0142] Performance data:

[0143] (1) According to the transcriptome enrichment results in Example 6, the PI3K / AKT signaling pathway has a significant effect on long-term cognitive impairment caused by ionizing radiation. In this example, the expression levels of genes related to the PI3K / AKT / GSK3β signaling pathway in the transcriptome were first statistically analyzed. Fig.19 As shown in A, compared with the blank control group, the GSK3β gene in the irradiated model group was upregulated, and the GPCR, Gβγ, PI3K, and AKT genes were downregulated; after intervention with Cistanche deserticola phenylethanoid glycosides, the GPCR, Gβγ, PI3K, and AKT genes were upregulated, and the GSK3β gene was downregulated.

[0144] Among them, the GSK3β gene induces neuronal cell damage and apoptosis, promotes the production of inflammatory factors, and further affects learning and memory functions. In addition, the GSK3β gene, as a key kinase in Tau protein phosphorylation, can directly or indirectly promote Tau protein hyperphosphorylation, thereby leading to neuronal cell pathology. Therefore, this example uses the GSK3β gene molecule as a clue to detect the content of inflammatory factors and cognitive-related proteins in the serum of mice in the blank control group (CON), irradiation model group (Mod), low-dose group (L), medium-dose group (M) and high-dose group (H) in Example 2. The results are as follows: Fig.19 As shown in BC, compared with the blank control group, the contents of IL-6 and TNF-α inflammatory factors in the irradiated model group were significantly increased (P < 0.05); after the intervention of Cistanche deserticola phenylethanoid glycosides, the serum levels of IL-6 and TNF-α inflammatory factors were significantly reduced in a dose-dependent manner, and the high-dose group had the most significant reduction effect (P < 0.05).

[0145] Since the GSK3β gene can directly or indirectly activate mouse cognition-related proteins, this example verifies mouse cognition-related markers. Fig. 20 As shown in the figure, compared with the blank control group, the relative expression of BDNF protein in the irradiated model group was decreased, and the phosphorylation level of Tau protein was increased; compared with the irradiated model group, the relative expression of BDNF protein in the low-dose group (L), medium-dose group (M) and high-dose group (H) after intervention with Cistanche deserticola phenylethanoid glycosides was increased, and the phosphorylation level of Tau protein was decreased, and the differences were significant (P < 0.05).

[0146] (2) Detection of oxidative stress indicators in mouse brain tissue

[0147] The increase of ROS will lead to an increase in oxidative stress response in animals. If the body's antioxidant system is damaged at the same time and cannot clear ROS in time, it will lead to a series of diseases. Previous experimental results have shown that the delayed reaction to ionizing radiation will still lead to an increase in the content of oxidative metabolites and a decrease in the content of antioxidant enzymes in the brain tissue of mice.

[0148] Since ionizing radiation activates cellular oxidative stress, which further leads to cognitive impairment, it is mainly caused by the accumulation of oxidative stress products ROS and the functional impairment of related antioxidant enzymes. Based on the transcriptome sequencing results in Example 6, this example uses protein imprint hybridization experiments and enzyme-linked immunosorbent assay experiments to verify the results of total glycosides of Cistanche deserticola reversing ionizing radiation-induced oxidative stress-related functions in mouse hippocampus tissue.

[0149] The results are as follows Fig.21 As shown in the figure, compared with the blank control group, the contents of ROS produced by peroxidation and MDA generated by lipid peroxidation in the brain tissue of mice in the irradiated model group were significantly increased, while the content of antioxidant GSH-px was significantly decreased (P < 0.05); compared with the irradiated model group, the contents of ROS and MDA in the low-dose group (L), medium-dose group (M) and high-dose group (H) after intervention with total glycosides of Cistanche deserticola were significantly decreased, among which, the contents of ROS and MDA in the medium-dose group (M) and high-dose group (H) were significantly decreased; the contents of GSH-px in the low-dose group (L), medium-dose group (M) and high-dose group (H) after intervention with total glycosides of Cistanche deserticola were significantly increased in the medium and high dose groups (P < 0.05).

[0150] (3) Detection of apoptosis-related protein expression in mouse hippocampus tissue

[0151] Based on the transcriptome sequencing results in Example 6, this example conducted a study on the effect of Cistanche deserticola phenylethanoid glycosides on cell apoptosis caused by ionizing radiation. Fig. 22 As shown in A, compared with the blank control group, the apoptosis-related genes bax, Casp3, Casp9 and p53 in the irradiated model group showed up-regulated expression after ionizing radiation treatment, while the anti-apoptosis gene bcl2 showed down-regulated expression after ionizing radiation treatment; after intervention with Cistanche deserticola phenylethanoid glycosides, the expression of bax, Casp3, Casp9 and p53 genes was significantly down-regulated, and the expression of anti-apoptosis gene bcl2 was significantly up-regulated in the low-dose group (L), medium-dose group (M) and high-dose group (H).

[0152] Western blot results Fig. 22 As shown in B, the verification results at the protein level are as follows Fig. 22 As shown in C, compared with the blank control group, the relative expression levels of apoptosis-related proteins p53, caspase3 and bax in the irradiated model group were significantly increased, while the expression of anti-apoptosis protein bcl2 was significantly decreased (P < 0.05); compared with the irradiated model group, the relative expression levels of apoptosis-related proteins p53, caspase3 and bax in the low-dose group (L), medium-dose group (M) and high-dose group (H) after intervention with total glycosides of Cistanche deserticola were decreased, while the relative expression level of anti-apoptosis protein bcl2 was significantly increased (P < 0.05).

[0153] In summary, ionizing radiation activates apoptosis-related signaling pathways in mouse brain tissue, leading to neuronal cell apoptosis, and then exhibiting symptoms related to cognitive impairment. The apoptotic response in the brain tissue of mice after intervention with the Cistanche phenylethanoid glycosides provided by the present invention decreased, indicating that the Cistanche phenylethanoid glycosides can inhibit neuronal cell apoptosis and thus exhibit a protective effect on brain tissue.

[0154] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. The use of Cistanche deserticola phenylethanoid glycosides in the preparation of a neuroprotective drug for treating long-term radiation damage, characterized in that: The long-term damage is caused by ionizing radiation.

2. A neuroprotective drug for treating late radiation damage, characterized in that: The medicine comprises Cistanche deserticola phenylethanoid glycoside and pharmaceutically acceptable auxiliary materials.

3. The drug according to claim 2, characterized in that The long-term damage is caused by ionizing radiation.

4. The drug according to claim 2, characterized in that The effective dosage of the Cistanche deserticola phenylethanoid glycoside is 100-200 mg / kg / day.

5. The drug according to claim 2, characterized in that The drug dosage form is any one of a gavage preparation, a tablet, a capsule or a liquid preparation.

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