Natural compound for preventing and treating radiation enteritis

Abstract

The invention discloses a natural compound for preventing and treating radiation enteritis, and belongs to the field of natural medicines. In particular to a new application of a natural compound lycopene in preparation of medicines for preventing and / or treating radiation enteritis. Cell experiments and mouse model in-vivo experiments prove that the lycopene can also significantly inhibit intestinal fibrosis. The invention reveals that lycopene is used as an active ingredient of the medicine for preventing and / or treating radiation enteritis, gives full play to the advantage of small toxic and side effects of natural compounds from plants, and provides valuable theoretical basis and reference for clinical medication of radiation enteritis.

Description

Technical Field

[0001] The invention relates to the field of natural medicines, and in particular to a natural compound for preventing and treating radiation enteritis. Background Art

[0002] At present, with the increasing use of nuclear-related technologies, more and more people are exposed to radiation passively or actively in various situations. First, a large number of people are suffering from ionizing radiation (IR) in nuclear medicine applications (such as X-rays, CT, radiotherapy, PET-CT, etc.) and nuclear industry safety accidents, especially nuclear accidents, and the harm to people will last for decades or even hundreds of years. Secondly, radiation from the environment is also an issue that cannot be ignored, such as electromagnetic radiation, radon (Rn) radiation in concrete or ore, and space radiation. At present, many safety measures have been established in radiation-related workplaces. However, due to lack of relevant training, improper operation and outdated facilities, there is still a possibility that technicians and radiation workers engaged in radiation medicine are exposed to ionizing radiation in the workplace that exceeds the safe dose for the human body. Therefore, people are exposed to radiation in many situations, and its impact is everywhere.

[0003] The intestine is one of the most radiation-sensitive organs in the human body. In radiotherapy for tumors in the abdominal or pelvic areas, radiation enteritis is very common because the treatment area is large and intestinal stem cells are extremely sensitive to radiation. In clinical diagnosis and treatment, about 50%-70% of patients who receive abdominal or pelvic radiotherapy develop acute radiation enteritis. Some patients even develop severe radiotherapy side effects such as intestinal contracture and intestinal fibrosis that progress to complete intestinal obstruction several years after treatment. Patients often undergo surgical fistula creation to improve obstruction symptoms. The permanent artificial anus after surgery brings varying degrees of stigma to patients, which seriously affects their quality of life. In addition, radiation side effects such as radiation enteritis severely limit the effective radiation dose used for radical radiotherapy.

[0004] Unfortunately, the pathogenesis of radiation-induced intestinal damage remains unclear. However, some studies still believe that acute radiation inflammation and chronic intestinal fibrosis induced by ionizing radiation are usually triggered by a series of biological processes, including the generation of reactive oxygen species (ROS), destruction of the antioxidant defense system, DNA damage, lipid peroxidation, and cell apoptosis. The acute inflammatory response of radiation to the intestine is mainly attributed to the inhibition of mitosis of intestinal stem cells in the crypts, which often leads to impaired regeneration of intestinal mucosal epithelial cells and loss of the mucosal barrier. Therefore, reducing the body's redox reaction and inhibiting the inflammatory factor storm are one of the main goals of the prevention and treatment strategy of radiation enteritis. Although synthetic compounds such as Amifostine have been shown to have effective radiation protection and can significantly reduce radiation damage caused by ionizing radiation, their use may still cause serious side effects including nausea, vomiting, and hypotension. Therefore, it is urgent to find natural ingredients as potential alternatives in order to provide safer radiation protection solutions. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a new use of lycopene, a natural compound, for preventing and treating radiation enteritis. Specifically, the use of lycopene in the preparation of a drug for preventing and / or treating radiation enteritis. The present invention verifies through in vivo experiments on mouse models that lycopene has no toxic effect on cells and is highly safe to use.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The invention discloses application of lycopene in preparing medicine for preventing and / or treating radiation enteritis.

[0008] Furthermore, the medicine is prepared with lycopene as the only active ingredient and supplemented with auxiliary materials used in medicines.

[0009] Furthermore, the dosage form of the drug includes tablets, capsules, powders, pills, granules, injections and patches.

[0010] The present invention also discloses a pharmaceutical composition for preventing and / or treating radiation enteritis, characterized in that the composition comprises lycopene.

[0011] Furthermore, the pharmaceutical composition also includes pharmaceutical excipients.

[0012] Furthermore, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculants, filter aids and release retardants.

[0013] Furthermore, the pharmaceutical composition includes a pharmaceutical carrier.

[0014] Furthermore, the drug carrier includes nanoparticles and liposomes.

[0015] Furthermore, the dosage forms of the pharmaceutical composition include injection, lyophilized powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] Lycopene, molecular formula C 40 H 56 , is a natural compound extracted from tomato fruit, without artificial synthetic ingredients and drug ingredients, non-toxic and harmless, and certified by the US Food and Drug Administration. It has strong antioxidant capacity and is very friendly to male prostate. As a fat-soluble carotenoid, it is the main pigment in ripe tomato fruit. The structure has 11 conjugated double bonds and two non-conjugated double bonds, which gives it excellent free radical scavenging ability and singlet oxygen quenching ability.

[0018] The present invention provides a new use of lycopene, specifically the use of lycopene in the preparation of a drug for preventing and / or treating radiation enteritis. The present invention verifies through in vivo experiments on mouse models that lycopene has no toxic effect on cells, does not affect the cell cycle and apoptosis process, and has high safety in use; lycopene can also significantly inhibit intestinal fibrosis. It reveals that lycopene is an effective ingredient for preventing and / or treating radiation enteritis, and gives full play to the advantages of plant-derived natural compounds with low toxicity and side effects, providing valuable theoretical basis and reference for clinical medication of radiation enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flow chart of animal experiments.

[0020] Figure 2 It is the length measurement of the colon from the ileocecal junction to the anal verge. A is a general picture of the intestine; B is the measurement of the length of the rectum (the distance from the colon to the anus).

[0021] Figure 3 The body weight of mice.

[0022] Figure 4 For histopathology and immunohistochemistry, A is the result of HE staining; B is the result of masson staining; C is the result of Sirius red staining; and D is the result of α-SMA immunohistochemistry staining.

[0023] Figure 5 is the effect of ionizing radiation on the body's state. Among them, A is the effect of lycopene on oxidative stress damage after irradiation; B is the effect of lycopene on liver and kidney damage induced by ionizing radiation.

[0024] Figure 6 Lycopene has anti-inflammatory effects.

[0025] Figure 7 The results of Western-blot detection of epithelial cell apoptosis, where A is mouse intestinal epithelial cell apoptosis; B is normal human epithelial cell apoptosis. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0027] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0028] Embodiment 1. Technical effect test materials and methods.

[0029] 1. Animal experiments.

[0030] SPF grade C57BL / 6 male mice, aged 6 weeks, weighing (18 g-20 g). Mice and standard feeding feed were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). All mice were housed in a controlled environment that met SPF standards. Before the formal start of the experiment, they were first adapted for one week. The feeding conditions of mice were set as follows: temperature 21-23 °C, humidity 0.5%, relative humidity 35%-65%, and light-dark cycle of 12 h to simulate diurnal changes. During the feeding period, mice had free access to water and food. All experimental protocols were in accordance with the requirements of the National Institutes of Health guidelines and were approved by the Animal Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences and were conducted in accordance with the guidelines of the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences and the National Guidelines for Laboratory Animal Welfare.

[0031] 2. Group management.

[0032] Experimental mice were randomly divided into the following four groups (6 mice in each group): control group (Con), enteritis model group (IR), LYC control group (10 mg / kg / d) (LYC), and LYC treatment group (10 mg / kg / d) (LYC+IR). Lycopene was dissolved in corn oil. The LYC group and the LYC+IR group were gavaged with 0.2 mL, 10 mg / kg of lycopene every day, and the remaining two groups were gavaged with the same amount of corn oil for three days. On the fourth day, the IR group and the IR+LYC group were irradiated with 12 Gy of ionizing radiation to induce radiation enteritis and continued gavage for one week after radiation. The survival and weight changes of mice were recorded throughout the experiment. 24 hours after the last administration, the feces of the mice were collected, and then the mice were killed, blood was collected from the eyeballs, serum was collected, the colon was removed and photographed, and the length of the colon from the ileocecal junction to the anal verge was measured. A small part of the intestine was fixed with 4% paraformaldehyde (PFA; #158127, Sigma) overnight for histopathological and immunohistochemical studies. The remaining intestines were washed with DEPC water, quickly frozen in liquid nitrogen and stored at -80°C. Figure 1 shown.

[0033] The dose of lycopene used was 10 mg / kg, which is equivalent to 56.8 mg / day for humans after adjusting for differences in body surface area using the allometric method. This is consistent with the risk assessment of lycopene conducted by the Council for Responsible Nutrition (USA) in 2006, which stated that the "Observed Safe Level (OSL)" of lycopene is within the range of 75 mg / d.

[0034] 3. Histopathological examination.

[0035] The mice were killed on the seventh day after irradiation. We first photographed the intestines of the mice and measured the length of the intestines. The intestinal tissues were fixed with 4% paraformaldehyde overnight and then graded for paraffin embedding according to standard procedures. They were stained with HE (hematoxylin and eosin), Masson staining, and Sirius red. The H&E-stained colon sections were evaluated histologically and graded as follows: (a) Inflammation severity, none = 0; mild = 1; moderate = 2; severe = 3; (b) Crypt damage, none = 0; basal 1 / 3 damage = 1; basal 2 / 3 damage = 2; only the surface epithelium = 1; basal 2 / 3 damage = 2; only the surface epithelium intact = 3; loss of the entire crypt and epithelium = 4; (c) Lesion site, none = 0; submucosal layer = 1; supramucosa = 2; muscular layer = 3; transmural = 4; The histological score is shown as the sum of the three evaluations, with the highest score being 11.22.

[0036] 4. Determination of intestinal function and oxidative stress indicators.

[0037] Blood samples were centrifuged at 4°C to collect serum. ALT (C009-3-1), AST (C010-3-1), creatinine (Cr) (C011-2-1), and blood urea nitrogen (BUN) (C013-2-1) in serum were measured using commercial kits provided by Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China) according to the manufacturer's instructions to evaluate the overall status. In addition, we also measured the content of malondialdehyde (MDA) (A003-1-2) and peroxidase (SOD) (A001-3-2) in the intestinal tissue of mice, as well as the activities of total antioxidant capacity (T-AOC) (A015-2-1) and glutathione peroxidase (GSH-PX) (A005-1-2) to reflect the level of oxidative stress.

[0038] 5. Immunohistochemistry.

[0039] After fixation with 4% paraformaldehyde overnight, the intestinal tissue was embedded in paraffin, sliced, and dewaxed with xylene. 5µm thick intestinal tissue sections were rehydrated in graded alcohol. Incubated with 10% goat serum at room temperature for 30min. Then incubated with α-SMA (1:200) primary antibody in a 4℃ refrigerator overnight. The sections were incubated with the same secondary antibody labeled with HRP primary antibody for 60min, then rinsed with TBST and 100µL of freshly prepared DAB was added, observed under a microscope, and color development was terminated with tap water. Counterstained with hematoxylin, differentiated with hydrochloric acid alcohol, and blued with blueing solution. Dehydrated and sealed, photographed, and quantitatively analyzed.

[0040] 6. Cell culture, cytotoxicity assays and clone formation experiments.

[0041] Normal human colon epithelial cells NCM460 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. These cells have been proven to be free of mycoplasma contamination. The cells were cultured in DMEM (Gibco, USA) containing 10% FBS (Gibco, USA), 2mM L-glutamine (Biosharp, China), 100U / mL penicillin, and 100µg / mL streptomycin (Biosharp, China) at 37°C and 5% CO2. We first investigated whether lycopene is toxic to normal human intestinal epithelial cells HIEC-6. The cells were cultured at a density of 1000 cells / well in 96-well plates. After adherence, different concentrations of lycopene were added and cultured for 48h. A 10% CCK-8 (Biosharp) solution diluted in serum-free DMEM was added to each well and incubated at 37°C for 1h. The absorbance at a wavelength of 450nm was measured using a microplate reader (Biotek, MQX200, Winooski, VT, USA). Cells were seeded in 6-well plates at a density of 500-2000 cells / well depending on the radiation dose. Cells were cultured overnight, pretreated with or without 10µM LYC for 24h, and then irradiated with 0, 1, 2, 4, and 8Gy of X-rays. Cells were then cultured at 37°C for 1-2 weeks to form colonies. They were fixed, stained with crystal violet, and observed and counted under a microscope.

[0042] 7. Immunoblotting Analysis.

[0043] After washing the cultured cells with PBS, the cell samples were collected with a cell scraper and NETN lysis buffer (NETN+PI) containing phenylmethylsulfonyl fluoride (PMSF) protease inhibitors and phosphorylase inhibitors was added. The intestinal tissue was ground in liquid nitrogen and lysed with NETN+PI lysis buffer, ultrasonically disrupted, and total protein was extracted. The total protein kit was used for quantitative analysis. Subsequently, the proteins were separated by electrophoresis and transferred to a 0.22µm polyvinylidene fluoride (PVDF) membrane by electrotransfer. The separated proteins were labeled with primary antibodies and then incubated with secondary antibodies of the corresponding species. The membrane was incubated in blocking solution (5% skim milk powder) for 1h and then incubated with the indicated primary antibodies overnight at 4°C. We used antibodies from different companies for immunoblotting: BAX, Bcl-2 (Abcam, UK); Caspase-3, Caspase-9 (huamei, China), PARP-1, β-actin (Proteintech, China). Subsequently, we washed the membranes with TBST three times for 10 min each time, and then incubated the membranes with HRP-conjugated secondary antibodies (Proteintech, goat anti-rabbit or goat anti-mouse) at room temperature for 1.5 h. Finally, the membranes were washed with TBST three times for 10 min each time and visualized using enhanced chemiluminescence agent CL (NCM Biotech, China). Amersham ImageQuant™ 800 gel imaging system was used for image acquisition. Image analysis was performed using ImageJ software for density analysis, and relative protein expression = target protein gray value / β-actin gray value. We used ImageJ software for visualization and quantitative analysis of protein bands.

[0044] 8. RNA extraction, cDNA synthesis, and quantitative real-time PCR (qPCR).

[0045] Intestinal tissues were quickly ground in liquid nitrogen, and total RNA was extracted using a Trizol-free extraction kit (RC112-01, Vazyme, Nanjing, China) according to the manufacturer's protocol. cDNA was synthesized by reverse transcription using the HiScript II Reverse Transcriptase Kit (11300ES92, YEASEN, Shanghai) according to the instructions. RT-PCR primers were designed according to the NCBI rat mRNA sequence database (Table 1). After primer synthesis, the RT-PCR reaction system was prepared according to the instructions of the 2×SYBR Green qPCR Master Mix (11202ES08, YEASEN, Shanghai) kit. The final volume of each qPCR reaction was 20 µL. The running conditions were as follows: 95°C for 10 min, 95°C for 15 s, 60°C for 1 min, then 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. After each run, the melting curve of each PCR product was obtained to control primer dimers and gene-specific peaks. The internal control was ACTIN. The specificity of the PCR products was confirmed by melting curve analysis.

[0046] Table 1 PCR primer sequence list .

[0047] 3. Results

[0048] 1. First, we studied whether lycopene can prevent and treat radiation colitis induced by ionizing radiation. Mice in the control group and IR group were gavaged with corn oil for 10 consecutive days, and mice in the LYC group and LYC+IR group were gavaged with 10 mg / kg LYC dissolved in corn oil for 10 consecutive days. After the third day of gavage, the IR group and LYC+IR group were irradiated with 12 Gy of whole abdomen. In the model group, mice after ionizing radiation attack showed severe colitis, manifested as weight loss. Compared with the IR group, LYC treatment significantly reduced weight loss (e.g. Figure 3 IR usually leads to colon shortening in IR-challenged mice. Oral administration of LYC can effectively alleviate IR-induced colon shortening. These results suggest that lycopene intervention can prevent and improve radiation-induced colon damage.

[0049] 2. HE staining showed that the intestinal villus cell structure of mice in the normal group and LYC group was intact and showed no signs of lesions. The IR group showed obvious shedding of intestinal villus epithelial cells, villus atrophy, inflammatory cell infiltration, crypt distortion, and decreased number of intestinal crypts. In mice treated with LYC, most of these histopathological abnormalities were restored. These results indicate that LYC can alleviate the development and progression of colitis (e.g. Figure 4 as shown).

[0050] 3. Evaluate the effect of ionizing radiation on the overall state of the body. Serum ALT and AST are two important indicators of liver function. Compared with the control group, the levels of ALT and AST in the serum of the IR group were significantly increased (P<0.0001), indicating that exposure to abdominal ionizing radiation caused liver damage. The ALT and AST levels in the liver of the LYC+IR group were significantly lower than those in the IR group (P<0.01), indicating that LYC can effectively alleviate the liver damage induced by ionizing radiation (such as Figure 5 B). SCr and BUN are the most commonly used indicators of renal injury. The levels of Cr and BUN in the serum of mice in the IR group were significantly higher than those in the control group, while the levels of Cr and BUN in the serum of mice in the LYC group were significantly lower than those in the control group. These results indicate that LYC can effectively alleviate liver and kidney damage induced by ionizing radiation.

[0051] To evaluate the effect of lycopene on oxidative stress in the body after irradiation, serum T-AOC, T-SOD, MDA, and GSH-PX levels were detected using commercial kits. Compared with the IR group, the increase in MDA levels in mice induced by ionizing radiation was largely attenuated by lycopene. In addition, compared with the normal control group, ionizing radiation caused a significant decrease in GSH, SOD, and T-AOC levels in the body after 7 days, while pretreatment with lycopene significantly restored the levels of GSH, SOD, and T-AOC in the intestinal tissues of mice, thereby protecting mice from acute radiation damage. These results indicate that lycopene can effectively alleviate the overall state of IR mice and reduce oxidative stress damage (such as Figure 5 A).

[0052] 4. The release of cytokines plays an important role in the development of radiation tissue damage. When an inflammatory response occurs in the intestine, the expression of inflammatory factors in the intestine will increase. Radiation-induced intestinal contracture and fibrosis may be caused by the continued occurrence of inflammatory responses. In order to explore whether lycopene has anti-inflammatory effects, we used RT-PCR to detect the expression levels of inflammatory factors such as TNF-α, IL-6, IL-1β, MCP-1, CXCL-1, CXCL2, CXCL10 and CD68 in intestinal tissues. The results of RT-PCR analysis showed that the expression of TNF-α, IL-6, IL-1β, MCP-1, CXCL-1, CXCL2, CXCL10 and CD68 in the intestine of model mice increased compared with the control group. At the same time, LYC treatment significantly reduced the expression of these genes in intestinal tissues. Therefore, LYC can improve the inflammatory response caused by ionizing radiation and alleviate the progression of radiation enteritis.

[0053] 5. Western-blot results showed that compared with the control group, the expression of Bax, Cleaved-Caspase3, and Cleaved-Parp-1 in the IR group increased, while the expression of Bcl-2 and Caspase-3 decreased. In addition, the expression of these proteins could be reversed after LYC intervention. These data indicate that ionizing radiation may cause cell damage through the apoptosis pathway, and the application of LYC may improve radiation enteritis (such as Figure 7 as shown).

[0054] In summary, the present invention provides a new use of lycopene, specifically the use of lycopene in the preparation of drugs for preventing and / or treating radiation enteritis. The present invention verifies through in vivo experiments on mouse models that lycopene has no toxic effect on cells, does not affect the cell cycle and apoptosis process, and has high safety in use; lycopene can also significantly inhibit colon shortening. Lycopene is revealed as an effective ingredient for preventing and / or treating radiation enteritis, and the advantages of natural compounds from plants with low toxicity and side effects are fully utilized, providing valuable theoretical basis and reference for clinical medication of radiation enteritis.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of lycopene in the preparation of a drug for preventing and / or treating radiation enteritis.

2. The use according to claim 1, characterized in that: The medicine is prepared with lycopene as the only active ingredient and supplemented with auxiliary materials used in medicines.

3. The use according to claim 1, characterized in that: The dosage forms of the drug include tablets, capsules, powders, pills, granules, injections and patches.

4. A pharmaceutical composition for preventing and / or treating radiation enteritis, characterized in that: The composition includes lycopene.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition also includes pharmaceutical excipients.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids and release retardants.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition includes a pharmaceutical carrier.

8. The pharmaceutical composition according to claim 4, characterized in that The drug carriers include nanoparticles and liposomes.

9. The pharmaceutical composition according to any one of claims 4 to 8, characterized in that The dosage forms of the pharmaceutical composition include injection, freeze-dried powder for injection, suspension, implant, embolic agent, capsule, tablet, pill and oral solution.

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