CN116549431A - Application of Arachidonic Acid in the Preparation of Drugs for Preventing and Preventing Ionizing Radiation Damage - Google Patents

Abstract

The invention provides the application of arachidonic acid in the preparation of medicines for preventing and treating ionizing radiation damage, and belongs to the technical field of medicine. The arachidonic acid of the present invention can significantly improve the survival rate and weight loss of mice after X-ray ionizing radiation, damage to spleen and small intestine, and increase of inflammation level. The specific performance is that the experimental mice have no abnormal reaction, the survival rate is significantly increased, the body weight has recovered, the spleen weight has recovered, the hematopoietic function has recovered, the intestinal regeneration ability has been improved, the level of intestinal inflammation has been significantly reduced, and its own toxic side effects are low. It shows that arachidonic acid has a broad application prospect in alleviating ionizing radiation damage.

Description

Application of Arachidonic Acid in the Preparation of Drugs for Preventing and Preventing Ionizing Radiation Damage

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of arachidonic acid in the preparation of medicines for preventing and treating ionizing radiation damage.

Background technique

Ionizing radiation is a general term for X-rays, α, β charged particles, γ-rays and ultraviolet rays that easily ionize substances. There are two major sources of ionizing radiation, one is natural radiation (mainly cosmic and surface radiation), and the other is artificial radiation (mainly used in military, economic, and medical fields). With the deepening of people's research on ionizing radiation technology, it also brings potential nuclear radiation hazards to the world today while benefiting mankind. The wide application of radiotherapy in the medical field kills tumor cells and also causes local and (or) systemic adverse reactions, the most common is damage to the hematopoietic system and digestive system. Therefore, how to prevent and treat body damage caused by ionizing radiation has become a major concern worldwide.

Arachidonic acid (AA), whose system name is 5,8,11,14-all-cis-eicosatetraenoic acid, is the most abundant polyunsaturated fatty acid in human tissues in the omega-6 series . Arachidonic acid is the precursor of many kinds of eicosan derivatives, so it and its metabolites are widely involved in many aspects of human metabolism and play a role, especially in regulating inflammation and promoting regeneration. Ionizing radiation can directly act on DNA molecules to cause damage to DNA molecules and lead to breakage, and can also indirectly cause water molecules to generate a large number of oxygen free radicals, thereby activating various intracellular apoptosis signaling pathways and leading to cell apoptosis. So far, no application of arachidonic acid in ionizing radiation protection has been found.

Contents of the invention

The purpose of the present invention is to provide the application of arachidonic acid in the preparation of medicines for preventing and treating ionizing radiation damage. Arachidonic acid can significantly improve the survival rate and weight loss of mice after X-ray ionizing radiation and the damage of spleen and small intestine and the level of inflammation. Elevated, significant curative effect, less toxic and side effects.

In order to solve the problems of the technologies described above, the present invention provides the following technical solutions:

The invention provides the application of arachidonic acid in the preparation of X-ray ionizing radiation damage medicine.

Preferably, the arachidonic acid can significantly improve the survival rate and weight loss of mice after ionizing radiation, damage to spleen and small intestine, and increase in inflammation level.

Preferably, the X-ray radiation intervention dose is 4-8 Gy.

Preferably, the arachidonic acid is administered by intragastric administration.

Preferably, the dosage of the arachidonic acid is 5-15 mg/time, and it is administered 36-60 hours before the ionizing radiation.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention firstly applies arachidonic acid in the prevention and treatment of X-ray ionizing radiation damage. According to the experimental results of mice, administration of 0.2 mL of arachidonic acid suspension 2 days before irradiation can significantly reduce the risk of 6Gy X-ray damage. It can reduce the survival rate and body weight of mice, and can also significantly reduce the damage of 4GyX rays to the spleen and small intestine and reduce the level of inflammation, without causing drug damage to mice. X-ray ionizing radiation damage has broad application prospects.

Description of drawings

Figure 1 Changes in the survival rate of mice under high-dose radiation (6Gy) after arachidonic acid treatment (*p<0.05).

Fig. 2 Body weight changes of mice under high-dose radiation (6Gy) after arachidonic acid treatment.

Figure 3 Changes in spleen size (left) and weight (right) of mice exposed to moderate doses of radiation (4Gy) after arachidonic acid treatment (*p<0.05; **p<0.01).

Figure 4 Hematoxylin-eosin staining of spleen sections of mice irradiated at a moderate dose (4Gy) after arachidonic acid treatment.

Figure 5 Periodic acid Schiff staining of small intestine sections of mice irradiated at a moderate dose (4Gy) after arachidonic acid treatment.

Fig. 6 After arachidonic acid treatment, the expression levels of inflammatory factors were detected by enzyme-linked immunoassay (left 1, 2) and quantitative PCR (right 1, 2) of small intestine tissue of mice under medium dose radiation (4Gy) (*p< 0.05).

Detailed ways

The invention provides the application of arachidonic acid in the preparation of ionizing radiation damage medicine. The arachidonic acid system of the present invention is called 5,8,11,14-trans-eicosatetraenoic acid, which is an important polyunsaturated fatty acid of the omega-6 series; the CAS registration number is 506- 32-1; chemical formula is C ₂₀ H ₃₂ O ₂ ; molecular weight is 304.467; melting point is -49°C; boiling point is 407.5°C; flash point is 336.3°C; density is 0.922g/cm ³ ; logP is 6.91; refractive index is 1.501 ; Appearance is colorless to light yellow oily liquid; Solubility is soluble in ethanol, acetone, benzene and other organic solvents, insoluble in water.

In the present invention, the arachidonic acid can significantly improve the survival rate and weight loss of mice after ionizing radiation, damage to spleen and small intestine, and increase in inflammation level. In the present invention, arachidonic acid is applied 2 days before X-ray radiation. After ionizing radiation, the mice do not show abnormal reaction, the survival rate increases significantly, the body weight rises, the spleen weight rises, the hematopoietic function recovers, and the intestinal regeneration ability The level of intestinal inflammation was significantly reduced, and there was no drug toxicity to mice.

In the present invention, the ionizing radiation is X-ray radiation, the X-ray radiation intervention dose is 4-8Gy, preferably 4Gy or 6Gy, and the dose rate is 0.5-1.5Gy/min, preferably 1Gy/min. The X-ray radiation mode of the present invention is whole body uniform radiation.

In the present invention, the arachidonic acid is administered by intragastric administration. The dosage of arachidonic acid in the present invention is 5-15 mg/time, preferably 10 mg/time, and it is administered 36-60 hours before ionizing radiation, preferably 48 hours before. In the present invention, 2 days before the radiation, the mice are given intragastric intervention, and the next day, the mice are given 3 to 6 times in total, and the mice are subjected to X-ray ionizing radiation after the second gavage (the 0th day). The arachidonic acid described in the present invention needs to be formulated into an arachidonic acid suspension and applied to ionizing radiation damage. The concentration of the arachidonic acid suspension in the present invention is 45-55 mg/mL, preferably 50 mg/mL, and the dosage of the arachidonic acid suspension is 0.1-0.3 mL/piece, preferably 0.2 mL/piece.

In the present invention, the medicine includes arachidonic acid and pharmaceutical excipients that do not affect the efficacy of the arachidonic acid. The dosage form of the drug of the present invention includes, but not limited to, pills, tablets, granules, capsules, aerosols, sustained-release preparations and controlled-release preparations. The pharmaceutical excipients of the present invention include, but are not limited to, flavoring agents, excipients, fillers, binders, disintegrants, lubricants, solubilizers, bacteriostatic agents, and antioxidants.

In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

Embodiment 1 Arachidonic Acid Effects on Mouse Survival Rate and Body Weight Caused by X-ray Radiation

1. Preparation of arachidonic acid suspension (final concentration: 50mg/mL): Weigh an appropriate amount of arachidonic acid (Sigma, purity > 95%, product number: 10931-1G) into a centrifuge tube, and then add the corresponding volume normal saline (0.9% NaCl solution), put it into a pre-cooled ultrasonic machine and mix thoroughly to form a uniform suspension with a final concentration of 50mg/mL, store it in the dark at 4°C, and shake it up and down before gavage before use ( On the day of intragastric administration, it is used and formulated now).

2. Cultivation and grouping of experimental mice: 15 C57BL6 mice (all male, provided by Victoria Lihua Company) were raised under SPF conditions and divided into 5 mice in the arachidonic acid group (AA) and 5 mice in the 6Gy radiation group. One mouse (6Gy), 5 mice in the 6Gy radiation + arachidonic acid group (6Gy + AA), a total of 3 groups, the follow-up intervention experiment was carried out when the mice were 7 weeks old (at least one week of adaptation in the animal room).

3. Experimental steps:

(1) Two days before radiation, intragastric intervention was given to the above three groups. The AA and 6Gy+AA groups received 0.2 mL of arachidonic acid suspension per mouse; Stomach, a total of 5 times; (2) 6Gy and 6Gy+AA groups were given 6Gy whole-body uniform ionizing radiation after the second gavage (day 0); (3) starting from day 0, before gavage intervention Firstly, the survival rate of each group of mice was counted and the body weight was measured and recorded, and recorded every other day, a total of 9 times (up to the 16th day). The results are shown in Figures 1-2.

As shown in Figure 1, the survival rate of mice in the AA group was 100%, and the morphology was normal, which proved that the toxicity and side effects of arachidonic acid itself were low; the survival rate of the 6Gy+AA group was significantly higher than that of the 6Gy group (p<0.05), which proved that the peanut four Acenoic acid improves survival after high-dose ionizing radiation.

As shown in Figure 2, the weight of the mice in the AA group increased slowly, which proved that the toxicity of arachidonic acid itself was low; the body weight of the 6Gy+AA group was higher than that of the 6Gy group, which proved that arachidonic acid can increase the body weight after high-dose ionizing radiation.

Example 2 Effect of Arachidonic Acid on the Spleen, Intestine and Inflammation Levels of Mice Caused by X-ray Radiation

1. Cultivation and grouping of experimental mice: 15 C57BL6 mice (all males, provided by Victoria Lihua Company) were raised under SPF conditions and divided into 5 blank control groups (Con); 5 mice in the 4Gy radiation group ( 4Gy); 5 rats in the 4Gy radiation+arachidonic acid group (4Gy+AA), a total of 3 groups, and the follow-up intervention experiment was carried out when the mice were 7 weeks old (at least one week of adaptation in the animal room).

2. Experimental steps:

(1) 2 days before the X-ray radiation, the 4Gy group and the 4Gy+AA group were intragastrically intervened, the 4Gy group was normal saline 0.2mL/; the 4Gy+AA group was arachidonic acid suspension (embodiment 1 Prepared in step 1) 0.2mL/piece, gavaged every other day, 5 times in total; (2) after the second gavage (the 0th day), the 4Gy and 4Gy+AA groups were subjected to uniform ionizing radiation of 4Gy X-rays; (3 ) on the 8th day, the mice in the 3 groups were killed by decapitation, the spleen was collected, its size, shape and weight were observed, and then the hematoxylin-eosin staining method (H&E staining) was carried out; the small intestine tissue was collected, and periodic acid Schiff staining (AB-PAS staining), and extraction of small intestine tissue for enzyme-linked immunoassay assay: according to the mouse interleukin 6 (IL-6) enzyme-linked immunoassay (ELISA) kit instructions for determination and mouse malondialdehyde ( MDA) Enzyme-linked immunoassay (ELISA) instructions (both purchased from Shanghai Yuanchuang Biotechnology Co., Ltd.) determination, and quantitative PCR experiment: according to the universal dye method qPCR special master mix for stable detection of low expression system (purchased from Nanjing Nuowei Zan Biotechnology Co., Ltd.) instructions to implement the experiment, the primers involved were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. to detect the expression level of intestinal inflammatory factors, and the specific primer sequences are:

GAPDH-F (SEQ ID NO: 1): 5'-TGTTTCCTCGTCCCGTAGA-3';

GAPDH-R (SEQ ID NO:2): 5'-CAATCTCCACTTTGCCACTG-3';

IL-6-F (SEQ ID NO: 3): 5'-TAGTCCTTCCTACCCCAATTTCC-3';

IL-6-R (SEQ ID NO:4): 5'-TTGGTCCTTAGCCACTCCTTC-3';

TNFα-F (SEQ ID NO:5): 5'-CCCTCACACTCAGATCATCTTCT-3';

TNFα-R (SEQ ID NO: 6): 5'-GCTACGACGTGGGCTACAG-3'. The results are shown in Figures 3-6.

Among them, the H&E staining experimental steps are as follows:

(1) Sample fixation: the removed fresh spleen tissue was fixed in 10% formaldehyde fixative solution;

(2) Tissue embedding: Put the corresponding tissue into the embedding frame, mark it, dehydrate it, and embedding: 70% alcohol for 24 hours → 80% alcohol for 2 hours → 90% alcohol for 2 hours → 100% alcohol (1) for 30 minutes →100% alcohol (two) 30min→alcohol: xylene (1:1) 25min→xylene 25min→paraffin (one) 45min→paraffin (two) 45min→embedding

(3) Embedding & Sectioning: After embedding in paraffin, store the wax block at 4°C. Slicing: cut to a thickness of 4 μm, and place the tissue slices in a 65°C oven overnight;

(4) Dyeing: 37°C oven xylene (1) 15min→37°C oven xylene (2) 15min→100% alcohol (1) 5min→100% alcohol (2) 5min→90% alcohol 5min→80% alcohol 5min → 70% alcohol for 5 minutes → triple distilled water for 5 minutes × 2 times → hematoxylin for 5 minutes → running water for 10 minutes → 1% HCl for 4 times → running water for 10 minutes → water-soluble eosin for 5 minutes → 70% alcohol for 4 times → 80% alcohol Swing 4 times → 90% alcohol, swing 4 times → 100% alcohol (1) 1 min → 100% alcohol (2) 1 min → xylene (1) 15 min → xylene (2) 15 min;

(5) Mount the slide: mount the slide with a mounting agent (histomount), and place in an oven at 37°C overnight.

(6) Observation: Observe the section under a microscope, take pictures and save the image data to observe that the cytoplasm is pink and the nucleus is blue-purple.

The experimental steps of AB-PAS staining are as follows:

Solution preparation: (1) 0.3% Oxinland acetic acid solution: Oxinland 0.3g, distilled water 97mL, glacial acetic acid 3mL, (2) 1% periodic acid aqueous solution, schiff reagent and sulfurous acid flushing solution with the same method as above.

Staining method: (1) Small intestine tissue sections were washed with conventional dewaxed water, and then washed with distilled water;

(2) Dye in 0.3% or 1% Aoxinlan acetic acid solution for 10-30 minutes;

(3) Fully wash with distilled water, at least 4 times;

(4) 1% periodic acid aqueous solution oxidation 5-10 minute;

(5) wash with distilled water, at least 3-4 times;

(6) Schiff reagent staining for 10-30 minutes;

(7) Rinse directly with sulfurous acid for 3 times, each time for 2 minutes;

(8) Wash with running tap water for 5 minutes and then wash with distilled water;

(9) Hematoxylin stained nuclei, if necessary, fully decompose with hydrochloric acid and wash with tap water;

(10) 95% alcohol, dehydrated with absolute alcohol, transparent in xylene, and sealed with gum.

(11) Results: the acidic mucopolysaccharide was indigo blue, the neutral mucopolysaccharide was bright purple or magenta, and the mixture of neutral and acidic mucin was purple blue.

Figure 3 shows the spleen shape and weight. The size and weight of the spleen were significantly reduced after 4Gy dose of ionizing radiation, and the size and weight of the spleen were increased after arachidonic acid treatment.

As shown in the H&E staining of the spleen in Figure 4, the white pulp area of the spleen decreased significantly after 4Gy dose of X-ray ionizing radiation, and the congestion was severe.

As shown in the AB-PAS staining of the small intestine in Figure 5, the number of goblet cells in the small intestine decreased significantly after 4Gy dose of X-ray ionizing radiation, and the intestinal regeneration ability decreased. After arachidonic acid treatment, the number of goblet cells increased significantly, and the intestinal regeneration ability recovered.

As shown in Figure 6 by the detection of small intestinal inflammatory factors, the level of small intestinal inflammation significantly increased after 4Gy dose of X-ray ionizing radiation, and the level of inflammation decreased significantly after arachidonic acid treatment.

Based on the above experiments, it can be seen that after arachidonic acid treatment, the survival rate and body weight of mice subjected to X-ray ionizing radiation increased, the weight of the spleen increased, the shape of the spleen improved, the area of the white pulp of the spleen increased, and the congestion decreased, indicating that The hematopoietic function was improved, the regenerative ability of the small intestine was restored, and the levels of inflammatory factors in the small intestine were reduced, which indicated that arachidonic acid had the effect of significantly improving the injury of X-ray ionizing radiation mice.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (5)

1. The application of arachidonic acid in the preparation of medicines for preventing and treating X-ray ionizing radiation damage. 2 . The application according to claim 1 , wherein the arachidonic acid can significantly improve the survival rate and weight loss of mice after ionizing radiation, damage to the spleen and small intestine, and increase in inflammation level. 3. The application according to claim 1, characterized in that the X-ray radiation intervention dose is 4-8 Gy. 4. The application according to claim 1, characterized in that the arachidonic acid is administered by intragastric administration. 5. The application according to claim 1, characterized in that the dosage of said arachidonic acid is 5-15 mg/time, and it is administered 36-60 hours before ionizing radiation.