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

Abstract

The invention discloses application of cistanche phenylethanoid glycoside in preparation of a bone protection 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 bone protection medicine for treating radiation long-term injury. The number of porous vacuoles and void gaps at the cartilage are reduced, the number and area of osteoclasts are reduced, the number and density of bone trabecula are increased, and the bone mass at the interface of the distal femur cartilage is increased; the number and thickness of bone trabecula, the percentage of bone mass and the volume and density of femur are improved, and the distribution degree of the bone trabecula is reduced; the content of oxidative stress regulatory factors and anti-apoptosis reaction protein is increased, the content of apoptosis reaction protein is reduced, and osteoclast differentiation and bone matrix secretion are inhibited.

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 bone protection drugs for treating long-term radiation damage. Background Art

[0002] Ionizing radiation is widely used in scientific research, medicine, military industry and other fields. While it is widely used, it will also cause a certain degree of harm to human health. Therefore, people are gradually paying more attention to the degree of harm of ionizing radiation to human health after radiotherapy. Clinical studies have shown that ionizing radiation still has related damage several months later. This damage is called long-term damage of ionizing radiation.

[0003] Bones are important organs for human motor function. Once bones are severely damaged, the body's motor ability will decline and the quality of life will be directly damaged. Clinical studies have shown that cancer patients who have received radiotherapy have a significantly increased risk of fractures and bone loss. The main cause of bone loss after radiotherapy is damage to osteoblasts and bone blood vessels themselves and abnormal osteoclast function, which leads to bone metabolism damage. At present, it has been reported in the literature that the main reason for radiation-induced bone loss is that ionizing radiation induces osteoclast maturation and differentiation, significantly increases the number of osteoclasts in cancellous bone tissue, and accumulates bone loss. However, there is currently insufficient pharmacological research on the bone protection effect used to treat long-term damage caused by radiation. Therefore, it is of great clinical significance to explore new potential therapeutic drugs, anti-radiation therapy and drug development.

[0004] As a traditional Chinese medicine, Cistanche deserticola has the pharmacological effects of tonifying kidney yang and strengthening menstrual blood. According to TCM theory, the damage caused by ionizing radiation can be attributed to kidney deficiency. At the same time, kidney essence deficiency further leads to symptoms such as slow movement, akinesia, marrow damage, and memory loss. Cistanche deserticola phenylethanoid glycoside is the main active ingredient of Cistanche deserticola. Modern pharmacological studies have shown that it has pharmacological effects such as antioxidant effects.

[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 bone protection. Those skilled in the art are eager to develop a method for effectively treating long-term bone 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 bone damage caused by ionizing radiation, and provides the use of Cistanche deserticola phenylethanoid glycoside in the preparation of a bone protection 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 bone protection drugs for treating radiation-induced long-term damage, wherein the long-term damage is caused by ionizing radiation.

[0008] The second object of the present invention is to provide a bone protection drug for treating long-term 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 phenylethanoid glycosides provided by the present invention have a significant improvement effect on the poor arrangement and integrity of the distal femoral cartilage interface of mice caused by ionizing radiation, and the obvious porous vacuoles in the cartilage; after intervention with Cistanche phenylethanoid glycosides, the distal femoral cartilage interface of mice is tightly arranged, the number of trabeculae is increased, the number of porous vacuoles in the cartilage and the cavity space around the femur are reduced, and the number of osteoclasts and the area of ​​osteoclasts are reduced.

[0014] (2) The Cistanche phenylethanoid glycosides provided by the present invention have a significant improvement effect on the significant decrease in bone mass at the distal femoral cartilage interface of mice caused by ionizing radiation, the destruction of bone microstructure, the damage of trabecular structure, and femoral cavity; after intervention with Cistanche phenylethanoid glycosides, the bone mass at the distal femoral cartilage interface of mice was increased, and the number and density of trabecular structure were increased.

[0015] (3) The Cistanche deserticola phenylethanoid glycosides provided by the present invention have a significant improvement effect on the number of trabeculae, percentage of bone mass, femoral volume, femoral bone density and femoral surface density of mice caused by ionizing radiation, which are significantly reduced, and the distribution of trabeculae and bone surface area / volume ratio are slightly increased; after intervention with Cistanche deserticola phenylethanoid glycosides, the number of trabeculae, percentage of bone mass, femoral volume, femoral bone density, femoral surface density and trabecular thickness of mice were significantly increased, and the distribution of trabeculae in mice was reduced.

[0016] (4) After intervention with the Cistanche deserticola phenylethanoid glycosides provided by the present invention, the relative contents of oxidative stress regulatory factors HO-1 and Nrf2 in mice were increased, and the relative content of p21 was reduced; the relative expression levels of apoptosis response proteins p53 and caspase3 were reduced, and the relative expression level of anti-apoptosis response protein bcl2 was increased; the relative expression levels of RUNX2 protein and OPG protein were increased, and the relative expression level of RANKL protein was reduced. By activating the RUNX2 / RANKL pathway, the differentiation function of osteoclasts was inhibited, the secretion of bone matrix was inhibited, and the purpose of protecting bone tissue was achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The femoral tissue staining pathological picture of mice in Example 3; A is the HE staining pathological picture; B is the TRAP staining pathological picture;

[0018] Figure 2 The micro-CT scans of the femoral tissue of mice in Example 3; A is a frontal scan of the femoral tissue; B is a top-view scan of the femoral tissue;

[0019] Figure 3 Statistical graphs of related indicators of femoral tissue of mice in Example 3; A is a statistical graph of the number of trabeculae; B is a statistical graph of the percentage of bone mass; C is a statistical graph of femoral volume; D is a statistical graph of the distribution of trabeculae; E is a statistical graph of the bone surface area / volume ratio; F is a statistical graph of femoral bone density; G is a statistical graph of femoral surface density; H is a statistical graph of trabecular thickness;

[0020] Figure 4 : A is a graph showing the effect of phenylethanoid glycosides of Cistanche deserticola on the expression of oxidative stress-related proteins in femoral tissue of mice in Example 4; A is a graph showing the results of Western blot; B is a statistical graph showing the relative expression of proteins; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; # P<0.05, ## indicates P < 0.01, ### indicates P < 0.001;

[0021] Figure 5 : A is a graph showing the effect of phenylethanoid glycosides of Cistanche deserticola on the expression of apoptosis-related proteins in femoral tissue cells of mice in Example 4; A is a graph showing the results of Western blot; B is a statistical graph showing the relative expression of proteins;

[0022] Figure 6 Graph showing the effect of Cistanche deserticola phenylethanoid glycosides on the expression of bone metabolism marker-related proteins in mouse femoral tissue in Example 4; A is a graph showing Western blot results; and B is a statistical graph showing relative protein expression levels. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] Example 1: Preparation of Cistanche deserticola phenylethanoid glycoside aqueous solution

[0026] 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℃;

[0027] Preparation of Cistanche phenylethanoid glycoside aqueous solution: Calculate the average body weight of each group of mice and the total weekly dose. Prepare the Cistanche phenylethanoid glycoside aqueous solution immediately before administration, taking mice with an average body weight of 25g as an example;

[0028] Weigh 20 mg of Cistanche deserticola phenylethanoid glycoside powder on an electronic balance and transfer it to a 15 mL centrifuge tube. Add 10 mL of deionized water using a pipette and shake thoroughly. Mark it as a high dose.

[0029] 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;

[0030] 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;

[0031] 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.

[0032] Example 2: Preparation of a model of long-term damage caused by ionizing radiation

[0033] 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.

[0034] 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).

[0035] 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.

[0036] Example 3: Morphological observation of mouse femur tissue pathological sections

[0037] (1) Specimen acquisition: On the night before specimen collection, the mice in the normal control group (CON), irradiated model group (Mod), model group + low-dose Cistanche phenylethanoid glycoside administration group (L), model group + medium-dose Cistanche phenylethanoid glycoside administration group (M), and model group + high-dose Cistanche phenylethanoid glycoside administration group (H) prepared in Example 2 were deprived of water and food; specimens were collected the next day, and the femurs of mice in each group were separated using scissors, tweezers and sterile gauze, one part of which was immersed in tissue fixative, and the other part was stored in a -80°C refrigerator.

[0038] (2) HE staining of mouse femur tissue

[0039] S1: fix the mouse femoral tissue specimen obtained in Example (1) with 4% paraformaldehyde in an amount ten times the amount of the mouse femoral tissue to be tested for ≥48 h, place the fixed mouse femoral tissue in a decalcification solution, decalcify at a constant temperature of 37°C, change the decalcification solution 2-3 times a week, and regularly use a needle to gently poke the non-sampled area. If there is no resistance, it means that the mouse femoral tissue is decalcified; if there is resistance, the mouse femoral tissue still needs to be decalcified; when the mouse femoral tissue is decalcified, the next step of the experimental operation can be carried out;

[0040] S2: Take out the femoral tissue of the mouse to be tested that has been decalcified in S1 from the decalcification solution, and soak the femoral tissue slices of the mouse to be tested 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 three times with tap water;

[0041] S3: Immerse the femoral tissue slices of the mouse to be tested after washing in S2 in hematoxylin dye and completely immerse them for 1-3 minutes. After the staining is completed, take out the femoral tissue slices of the mouse to be tested, wash them with tap water for 3 times until there is no dye. After washing, use hydrochloric acid aqueous solution to differentiate them, and observe the differentiation state under a microscope. After the differentiation is completed, use distilled water to wash them for 3 times. After the differentiation is completed, immerse the femoral tissue slices of the mouse to be tested in ammonia aqueous solution to turn blue. After the blueing is completed, use running water to rinse off the excess dye;

[0042] S4: completely immerse the cleaned femoral tissue slices of the mouse to be tested in S3 into the eosin staining solution and fully stain for 2-3 minutes; sequentially place the stained femoral tissue slices of the mouse to be tested in xylene I and xylene II and soak them for 5 minutes respectively, observe the brain tissue slices until they are transparent, take out the bone tissue slices and evenly seal them with neutral gum, observe the brain tissue slices under an electron microscope, and perform image acquisition and analysis after scanning.

[0043] The results are as follows Figure 1As shown in A, the bone of the distal femoral cartilage interface of the blank control group (CON) mice was compact, the structure was full, and the cartilage morphology and structure were complete; the distal femoral cartilage interface of the irradiated model group (Mod) mice was not arranged, the integrity was poor, and obvious pore-like vacuoles appeared in the cartilage. Due to the decrease in the number and thickness of trabeculae, large cavities appeared in the distal femoral structure. Compared with the irradiated model group, the distal femoral cartilage interface of the mice in the medium-dose (M) and high-dose (H) Cistanche total glycosides intervention groups was closely arranged. Due to the increase in the number of trabeculae, the number of pore-like vacuoles in the cartilage was significantly reduced, and the cavity gap around the femur became smaller. The effect of the high-dose group was the most significant, while the consolidation of the tissue of the mice in the medium- and low-dose (L) Cistanche total glycosides intervention groups was not significantly different from that of the irradiated model group.

[0044] (3) Tartrate-resistant acid (TRAP) staining of mouse femur tissue

[0045] S1: fix the mouse femoral tissue specimen obtained in Example (1) with 4% paraformaldehyde in an amount ten times the amount of the mouse femoral tissue to be tested for ≥48 h, place the fixed mouse femoral tissue in a decalcification solution, decalcify at a constant temperature of 37°C, change the decalcification solution 2-3 times a week, and regularly use a needle to gently poke the non-sampled area. If there is no resistance, it means that the mouse femoral tissue is decalcified; if there is resistance, the mouse femoral tissue still needs to be decalcified; when the mouse femoral tissue is decalcified, the next step of the experimental operation can be carried out;

[0046] S2: Take out the femoral tissue of the mouse to be tested that has been decalcified in S1 from the decalcification solution, and soak the femoral tissue slices of the mouse to be tested 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 three times with tap water;

[0047] S3: Preparation of dye solution: Use 18 mL of solution A (acetic acid buffer), 1 mL of solution B (hexazo para-fuchsin) and 1 mL of solution C (naphthol AS-BI phospholipid solution) to prepare the incubation solution; fully mix the above reagents in a 50 mL centrifuge tube, add 0.282 g of potassium sodium tartrate, shake well to fully dissolve it, use a marker pen to mark it as TRAP incubation solution, and set it aside for later use;

[0048] S4: Add the TRAP incubation solution prepared in S3 to the femoral tissue slices of the mouse to be tested obtained in S2 until the incubation solution completely covers the femoral tissue slices of the mouse to be tested, incubate at room temperature for 1-2 hours, remove the bone tissue slices after incubation, wash them with distilled water for 3 times until there is no excess dye, and observe the bone tissue slices under a microscope.

[0049] TRAP staining shows that osteoclasts in bone tissue are wine red, irregular in shape, and mainly distributed at the edge of trabeculae, while bone marrow cells are dark blue. Figure 1 As shown in B, there were almost no burgundy cells at the interface of the distal femoral cartilage of the blank control group (CON) mice, while in contrast, obvious burgundy-positive cells were visible at the edge of the trabeculae at the distal femoral cartilage interface of the irradiated model group (Mod) mice, which were stained darker and showed an irregular distribution; compared with the irradiated model group, the number of burgundy-positive cells and the positive area in the Cistanche deserticola total glycosides intervention groups (low-dose group (L), medium-dose group (M) and high-dose group (H)) decreased, among which the decrease in the number of burgundy-positive cells and the positive area in the high-dose group (H) was particularly significant, while there was no significant difference in the positive cell area in the medium and low-dose groups (L) compared with the irradiated model group.

[0050] (4) Imaging scan of mouse femoral bone tissue

[0051] S1: μCT scanning: fix the mouse femoral tissue specimen obtained in Example (1) with 4% paraformaldehyde in an amount ten times the amount of the mouse femoral tissue to be tested for ≥48 h, place the fixed mouse femoral tissue to be tested on a scanning plane = perform Micro-CT scanning; set the scanning parameters as follows: X-ray tube current 200 μA, voltage 70 KV; obtain the original image of the mouse femoral tissue to be tested after scanning, perform ROI analysis on the mouse femur using CT Analyser (software version 1.20.3.0, Bruker, Germany) and calculate bone-related indices for subsequent statistical analysis;

[0052] S2: μCT reconstruction: The original image of the femoral tissue of the mouse to be tested obtained after the scanning in S1 was reconstructed in a specific area using the three-dimensional reconstruction software NRecon (software version V1.7.4.2, Bruker, Germany). First, the original image was previewed overall, and the areas to be observed, such as the distal end of the femur and the femoral cross section, were selected, and the reconstruction parameters were adjusted and set for image reconstruction;

[0053] S3: μCT analysis: CT Analyser (software version 1.20.3.0, Bruker, Germany) was used to analyze and record the indicators that needed to be statistically analyzed.

[0054] The results are as follows Figure 2As shown in the figure, compared with the blank control group (CON), the bone mass at the distal femoral cartilage interface of the irradiated model group (Mod) was significantly reduced, and the bone microstructure was significantly destroyed; compared with the irradiated model group, the Cistanche deserticola phenylethanoid glycoside intervention groups (low-dose group (L), medium-dose group (M) and high-dose group (H)) could increase the bone mass at the distal femoral cartilage interface of the mice to a certain extent, among which the medium-dose group (M) and high-dose group (H) had a more significant therapeutic effect on the destruction of bone microstructure caused by ionizing radiation, while the low-dose group (L) had a less obvious therapeutic effect on the destruction of bone microstructure caused by ionizing radiation. It can also be seen from the top scan of the femoral tissue that compared with the blank control group (CON), the irradiated model group mice had obvious damage to the trabecular structure and obvious femoral cavity; compared with the irradiated model group (Mod), the Cistanche deserticola intervention groups (low-dose group (L), medium-dose group (M) and high-dose group (H)) can increase the number and density of trabecular structure, among which the medium-dose group (M) and high-dose group (H) have the best effect on increasing the number and density of trabecular structure.

[0055] In this example, Graphpadprism 8.4.3 software was used to perform statistical analysis on the experimental results of micro-CT scanning of mouse femur tissue. Figure 3 As shown in the figure, compared with the blank control group (CON), the trabecular number, bone mass percentage, femoral volume, femoral bone density and femoral surface density of the irradiated model group (Mod) mice were significantly reduced, the trabecular thickness was slightly decreased, and the distribution of trabecular bone and the bone surface area / volume ratio were slightly increased, with no significant changes. Compared with the irradiated model group (Mod), the total glycosides of Cistanche deserticola intervention groups (low-dose group (L), medium-dose group (M) and high-dose group (H)) can increase the number of trabecular bone, bone mass percentage, femoral volume, femoral bone density, femoral surface density and trabecular bone thickness of mice, and reduce the distribution of trabecular bone and bone surface area / volume ratio. Among them, the high-dose group (H) can significantly increase the number of trabecular bone, bone mass percentage, femoral volume, femoral bone density, femoral surface density and trabecular bone thickness of mice, and significantly reduce the distribution of trabecular bone of mice.

[0056] Example 4: Detection of Mouse Femoral Tissue-Related Proteins

[0057] (1) Total protein extraction from mouse femur tissue

[0058] In this example, two femoral tissue specimens of mice in each group obtained in Example 3 were taken, placed in a -20°C precooled mortar, and ground with liquid nitrogen. After the mouse femoral tissue was ground into a powder state, it was quickly transferred to a 1.5mL EP tube and placed on ice. After all groups were ground, 200μL of lysis solution was added to each group, and lysis was performed on ice for 30 minutes. After lysis, the centrifuge tube was placed in a 4°C precooled centrifuge, centrifuged at 12000rpm for 10 minutes, and the protein supernatant was aspirated to obtain the total protein of the mouse femoral tissue to be tested.

[0059] (2) BCA protein quantification

[0060] 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 femoral tissue of each group of mice to be tested obtained in (1), and 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.

[0061] (3) Protein denaturation

[0062] Add 80 μL of the total protein extract of femoral 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.

[0063] (4) Electrophoresis-membrane transfer

[0064] Prepare the gel and electrophoresis solution in advance according to the instructions. Shake the total protein samples of the femoral tissue of each group of mice to be tested obtained in (3) before loading, and centrifuge again. According to the concentration of the BCA protein quantification result in (2), load 20 μg of the protein sample obtained in (3) into each loading well. 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, and start electrophoresis at a voltage of 80 V. When the markers of different protein molecular weights are completely separated, adjust the voltage to 100 V and continue the voltage. End the electrophoresis when the sample is tangent to the green edge of the lower layer of the electrophoresis tank.

[0065] 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.

[0066] (5) Blocking and incubation with primary antibody-secondary antibody-exposure and development

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Performance data:

[0072] (1) Detection of oxidative stress-related protein expression in mouse femoral tissue

[0073] The accumulation of reactive oxygen species and the decrease in the activity of antioxidant enzymes produced by oxidative stress are also key factors leading to decreased bone density. In order to explore the effect of total glycosides of Cistanche deserticola on radiation-induced oxidative stress in bone tissue, this example conducted a protein immunoblot hybridization experiment on the femoral tissues of each group of mice to be tested.

[0074] The results are as follows Figure 4 As shown in the figure, compared with the blank control group (CON), the relative expression of oxidative stress regulators Nrf2 and HO-1 in the femoral tissue of mice in the irradiated model group (Mod) was significantly reduced, while the relative expression of p21 was significantly increased. Compared with the irradiated model group (Mod), the medium-dose group (M) and high-dose group (H) of the total glycosides of Cistanche deserticola intervention group significantly increased the relative content of HO-1, enhanced its antioxidant effect, and to a certain extent also increased the relative content of Nrf2, and significantly reduced the relative content of p21, while the relative content of Nrf2, HO-1 and p21 in the low-dose group (L) did not change significantly compared with the model group.

[0075] (2) Detection of apoptosis-related protein expression in mouse femoral tissue

[0076] Cell apoptosis is also another factor that causes pathological changes in bone tissue. Ionizing radiation not only enhances the body's oxidative stress response, but also induces the body's cell apoptosis response. The two work together to cause the long-term effects of radiation. Therefore, in order to explore whether total glycosides of Cistanche deserticola have a therapeutic effect on the apoptosis response caused by ionizing radiation, this example conducted a protein imprint hybridization experiment of marker proteins related to apoptosis response.

[0077] The results are as follows Figure 5 As shown in the figure, compared with the blank control group (CON), the relative expression of apoptosis response proteins p53, caspase3 and bax in the femoral tissue of mice in the irradiated model group (Mod) was significantly increased, and the relative expression of anti-apoptosis response protein bcl2 was significantly decreased. Compared with the irradiated model group (Mod), the medium-dose group (M) and high-dose group (H) of the total glycosides of Cistanche deserticola intervention group can significantly reduce the relative expression of apoptosis response proteins p53 and caspase3, and there is no significant change in the relative expression of bax protein, while the relative expression of anti-apoptosis response protein bcl2 is slightly increased, but there is no significant difference; while the relative contents of p53, caspase3, bax and bcl2 proteins in the low-dose group (L) were not significantly changed compared with the model group.

[0078] (3) Detection of protein expression levels of metabolic markers in mouse femoral tissue

[0079] Ionizing radiation can cause the expression of osteoblasts in mouse bone tissue to reduce the content of related markers, and the content of osteoclast markers increases, thereby causing bone damage in mice. The bone metabolism process is regulated by a variety of secretory factors, so the bone loss caused by radiation is the result of a variety of signaling pathways co-regulated, among which RANK / RANKL / OPG is a classic bone metabolism pathway, and RANKL directly stimulates immature osteoblasts to differentiate into osteoclasts, which is a key factor in osteoclast maturation. OPG is osteoprotegerin, which further inhibits the differentiation of osteoclasts by inhibiting the combination of RANKL and RANK, and has an important protective effect on bone formation. The present embodiment is to explore whether the total glycosides of Cistanche deserticola can intervene in the changes in the content of bone-related markers caused by irradiation, and the expression of related proteins of metabolic markers of femoral tissue of each group of mice to be tested is detected.

[0080] The results are as follows Figure 6 As shown in the figure, compared with the blank control group (CON), the relative expression of RUNX2 protein and OPG protein in the femoral tissue of mice in the irradiated model group (Mod) was significantly decreased, and the relative expression of RANKL protein was significantly increased, which led to excessive differentiation of osteoclasts. Compared with the irradiated model group (Mod), the Cistanche deserticola total glycosides intervention groups (low-dose group (L), medium-dose group (M) and high-dose group (H)) increased the relative expression of RUNX2 protein and OPG protein, and decreased the relative expression of RANKL protein, inhibited the differentiation function of osteoclasts, and had a therapeutic effect on excessive bone resorption caused by radiation; among them, the high-dose group (H) significantly increased the relative expression of RUNX2 protein and OPG protein, and significantly decreased the relative expression of RANKL protein, which had a significant therapeutic effect on excessive bone resorption caused by radiation.

[0081] In summary, the Cistanche phenylethanoid glycosides provided by the present invention have the function of significantly increasing the bone volume, bone surface area density, bone percentage, bone density and the number of trabeculae in mice, and significantly increasing the relative expression levels of RUNX2 protein and OPG protein, reducing the relative expression level of RANKL protein, activating the RUNX2 / RANKL pathway, inhibiting the differentiation function of osteoclasts, inhibiting the secretion of bone matrix, and having a good protective effect on bone tissue.

[0082] 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 bone protection drug for treating long-term radiation damage, characterized in that: The long-term damage is caused by ionizing radiation.

2. A bone protection 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.

Images