Use of a lactic acid bacterium in the manufacture of a functional product for preventing and treating radiation enteritis

Abstract

The application belongs to the technical field of biological medicine, and discloses application of lactic acid bacteria in preparation of a functional product for preventing and treating radiation enteritis and a medicine for treating radiation enteritis through a PI3K-AKT-mTOR pathway, wherein the lactic acid bacteria is Lactobacillus plantarum CGMCC No.8198. The application finds application of a lactic acid bacteria in improving radiation enteritis. Experiments are conducted by using live Lactobacillus plantarum CGMCC No.8198 liquid, and the experiments show that the lactic acid bacteria can inhibit the increase of inflammatory factor levels in the intestinal tract caused by radiation, improve intestinal barrier damage caused by radiation, improve the disturbed intestinal flora structure, and inhibit PI3K-AKT-mTOR related factors significantly activated by radiation, so as to achieve the function of improving radiation enteritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular, it relates to the application of a type of lactic acid bacteria in the preparation of functional products for the prevention and treatment of radiation enteritis via the PI3K-AKT-mTOR pathway. Background Technology

[0002] Members of the genus Lactobacillus have long been considered one of the most abundant microorganisms in the human gastrointestinal tract and are important beneficial bacteria in the human gut. L. plantarum CGMCC8198 is a novel probiotic strain with independent intellectual property rights, isolated and screened by our research group in the previous stage. Besides exhibiting acid and bile salt resistance (patent number: 201310491340.4), heat resistance, and weight loss and lipid-lowering effects (patent number: 201310518617.8), it can also colonize the colon of mice and play a role in regulating the intestinal tract. However, there are currently no reports exploring the effects of this bacterium on intestinal diseases, and even fewer studies on how it improves the treatment of radiation enteritis.

[0003] Radiation enteritis is a common complication in patients with pelvic and abdominal tumors who undergo radiotherapy. This condition can prevent patients from receiving the full dose and duration of treatment, thus affecting their survival rate.

[0004] Clinically, the "Consensus Report on the Clinical Application of Probiotics" formulated by the Southeast Asian Gastric Nerve Motion Association Working Group in 2018 clearly states that probiotics can effectively reduce diarrhea caused by radiotherapy; and the "Chinese Expert Consensus on the Diagnosis and Treatment of Radiation Proctitis (2018 Edition)".

[0005] A 2022 article published in *Microbiome* (Q1, IF: 14.652), a top international microbiology journal, provided the first evidence that targeted intervention in the gut microbiota may be an effective means of preventing and treating intestinal damage. Furthermore, in August 2022, *Nature Reviews Endocrinology* (Q1, IF: 44.9) published an article titled "The role of ironin host-microbiota crosstalk and its effects on systemic glucose metabolism," which clearly elucidated the close relationship between intestinal inflammation and probiotics.

[0006] Therefore, research on probiotics that can improve the treatment of radiation enteritis, a postoperative complication in patients with pelvic and abdominal tumors, is of great significance to human health. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of lactic acid bacteria through the PI3K-AKT-mTOR pathway in the preparation of functional products for preventing and treating radiation enteritis.

[0008] The technical solution adopted by this invention to solve its technical problem is: The application of a lactic acid bacterium via the PI3K-AKT-mTOR pathway in the preparation of functional products for the prevention and treatment of radiation enteritis, wherein the lactic acid bacterium is *Lactobacillus plantarum* (…). Lactobacillus plantarum CGMCC No. 8198.

[0009] Furthermore, the Lactobacillus plantarum CGMCC No. 8198 was deposited on September 17, 2013, at the China General Microbiological Culture Collection Center (CGMCC), No. 1, Beichen West Road, Chaoyang District, Beijing, China.

[0010] Furthermore, the live bacterial solution of *Lactobacillus plantarum* CGMCC No. 8198 can inhibit inflammatory factors in the irradiated intestine.

[0011] Furthermore, the live bacterial solution of *Lactobacillus plantarum* CGMCC No. 8198 can improve radiation-induced intestinal barrier damage.

[0012] Furthermore, the live bacterial solution of *Lactobacillus plantarum* CGMCC No. 8198 can improve the gut microbiota structure disrupted by radiation.

[0013] Furthermore, the live bacterial culture of *Lactobacillus plantarum* CGMCC No. 8198 can significantly inhibit PI3K-AKT-mTOR-related factors.

[0014] Furthermore, the functional products include pharmaceuticals.

[0015] Furthermore, in this invention, after treatment with *Lactobacillus plantarum* CGMCC No. 8198, ELISA detection showed that the levels of inflammatory factors IL-1β and IL-6 were reduced.

[0016] Furthermore, in this invention, after treatment with *Lactobacillus plantarum* CGMCC No. 8198, HE and MASSON staining were performed to observe intestinal barrier damage, and it was found that it could improve radiation-induced intestinal barrier damage.

[0017] Furthermore, 16S rRNA analysis performed using *Lactobacillus plantarum* CGMCC No. 8198 in this invention revealed that it can improve the intestinal flora disorder caused by radiation.

[0018] Furthermore, in this invention, after treatment with *Lactobacillus plantarum* CGMCC No. 8198, the expression of factors such as PI3K and AKT was detected by Western blot, and it was found that it can inhibit the PI3K-AKT-mTOR pathway-related factors that are significantly activated by radiation.

[0019] The advantages and positive effects of this invention are as follows: 1. This invention discovers the application of a lactic acid bacterium in improving radiation enteritis. Experiments were conducted using live bacterial suspension of *Lactobacillus plantarum* CGMCC No. 8198. The experiments showed that it can inhibit the increase in the level of inflammatory factors in the intestine caused by radiation, improve radiation-induced intestinal barrier damage, improve the disordered intestinal flora structure, and inhibit PI3K-AKT-mTOR-related factors that are significantly activated by radiation, thereby achieving the function of improving radiation enteritis.

[0020] 2. This invention provides an application of lactic acid bacteria to treat and improve the condition through the PI3K-AKT-mTOR pathway. Experiments were conducted using live bacterial culture of *Lactobacillus plantarum* CGMCC No. 8198. The experiments showed that it can directly reduce the activity of genes such as IL-6, IL-1β, PI3K, and AKT in radiation enteritis cells, thus demonstrating its role in preventing and treating radiation enteritis.

[0021] 3. The strains used in this invention are food-grade probiotics, which are highly safe for conducting related drug research.

[0022] 4. This invention provides new theoretical guidance for the treatment of radiation enteritis.

[0023] 5. Experiments of this invention show that *Lactobacillus plantarum* can inhibit the increase in intestinal inflammatory factor levels caused by radiation, improve intestinal barrier damage, improve the disordered intestinal flora structure, and inhibit the levels of pathway-related factors, thereby improving radiation enteritis, a postoperative complication in patients with pelvic and abdominal tumors. *Lactobacillus plantarum* CGMCC No. 8198 of this invention can be used in the medical and other fields, and can be used to develop related drugs. Attached Figure Description

[0024] Figure 1 This is a graph showing the levels of inflammatory factors IL-1β and IL-6 detected by ELISA after treatment with *Lactobacillus plantarum* CGMCC No. 8198 in this invention. Figure 2 The images show the intestinal barrier damage observed after treatment with *Lactobacillus plantarum* CGMCC No. 8198 in this invention, followed by HE and MASSON staining. The first row of images shows HE staining, and the second row shows MASSON staining. Figure 3This is a diagram of 16S rRNA analysis performed using *Lactobacillus plantarum* CGMCC No. 8198 in this invention. Figure 4 This is a graph showing the expression of factors such as PI3K and AKT after treatment with *Lactobacillus plantarum* CGMCC No. 8198 in this invention, detected by Western blot. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0026] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0027] The application of a lactic acid bacterium via the PI3K-AKT-mTOR pathway in the preparation of functional products for the prevention and treatment of radiation enteritis, wherein the lactic acid bacterium is *Lactobacillus plantarum* (…). Lactobacillus plantarum CGMCC No. 8198.

[0028] Preferably, the Lactobacillus plantarum CGMCC No. 8198 was deposited on September 17, 2013 at the China General Microbiological Culture Collection Center (CGMCC), No. 1, Beichen West Road, Chaoyang District, Beijing, China.

[0029] Preferably, the live bacterial solution of *Lactobacillus plantarum* CGMCC No. 8198 can inhibit inflammatory factors in the irradiated intestine.

[0030] Preferably, the live bacterial solution of *Lactobacillus plantarum* CGMCC No. 8198 can improve radiation-induced intestinal barrier damage.

[0031] Preferably, the live bacterial solution of *Lactobacillus plantarum* CGMCC No. 8198 can improve the radiation-disrupted intestinal flora structure.

[0032] Preferably, the live bacterial culture of *Lactobacillus plantarum* CGMCC No. 8198 can significantly inhibit PI3K-AKT-mTOR-related factors.

[0033] Preferably, the functional product includes pharmaceuticals.

[0034] Preferably, in this invention, after treatment with *Lactobacillus plantarum* CGMCC No. 8198, ELISA detection showed that the levels of inflammatory factors IL-1β and IL-6 were reduced.

[0035] Preferably, in this invention, after treatment with *Lactobacillus plantarum* CGMCC No. 8198, HE and MASSON staining were performed to observe intestinal barrier damage, and it was found that it can improve radiation-induced intestinal barrier damage.

[0036] Preferably, the 16S rRNA analysis performed using *Lactobacillus plantarum* CGMCC No. 8198 in this invention showed that it can improve the intestinal flora structure disorder caused by radiation.

[0037] Preferably, in this invention, after treatment with *Lactobacillus plantarum* CGMCC No. 8198, the expression of factors such as PI3K and AKT was detected by Western blot, and it was found that it can inhibit the PI3K-AKT-mTOR pathway-related factors that are significantly activated by radiation.

[0038] Specifically, the following examples illustrate the technical solution of the present invention through ELISA, HE, MASSON, 16S rRNA analysis and Western blot experiments, respectively.

[0039] Example 1: Animal Grouping and Treatment Forty male C57BL / 6 mice aged 4-6 weeks were purchased from Beijing Sifefu Biotechnology Co., Ltd. (License: SCXK (Xiang) 2017-0006). Animals were randomly divided into a blank control group, a radiation enteritis group, a positive control group (mesalazine enema), and a low-dose *Lactobacillus plantarum* 8198 group (5 × 10⁶ mice). 7 CFU / ml), high-dose Lactobacillus plantarum 8198 group (5×10) 9 CFU / ml). Animals were housed in an SPF animal laboratory at 18℃–22℃. After 5 days of acclimatization, modeling was initiated (abdominal irradiation with high-energy gamma rays (12 Gy, 225 kV) at 1.2 Gy / min for 9 minutes and 50 seconds). After modeling, the positive control group, the low-dose *Lactobacillus plantarum* 8198 group, and the high-dose *Lactobacillus plantarum* 8198 group were administered via gavage (0.1 ml / 10 g mice; once daily) for 28 days. Simultaneously, the control group and the radiation enteritis group were administered 0.9% saline via gavage (0.1 ml / 10 g mice; once daily).

[0040] Example 2: ELISA detection of IL-1β and IL-6 inflammatory factor levels 1. Blood was collected from the eyeballs of C57BL / 6 mice. The serum samples were left to stand at room temperature for 30 minutes. The levels of IL-1β and IL-6 in the serum were measured according to the instructions of the Mouse IL-1β and IL-6 ELISA Kit.

[0041] 2. OD was determined using an ultraviolet spectrophotometer. 450 .

[0042] Test results as follows Figure 1 As shown, from Figure 1 As can be seen from the ELISA results of intestinal tissue pathology in mice, compared with the normal group, the level of inflammatory factor IL-1β in the model group was significantly increased by about 35 pg / ml, and the level of IL-6 was significantly increased by about 200 pg / ml. After treatment, compared with the model group, the level of IL-1β in the low-dose group decreased by about 10 pg / ml, and the level of IL-6 decreased by about 100 pg / ml. Compared with the model group, the level of IL-1β in the high-dose group decreased by about 20 pg / ml, and the level of IL-6 decreased by about 80 pg / ml. The decreasing trend was dose-dependent, and the changes in the levels of each indicator were significant after treatment (p < 0.0001).

[0043] Example 3: HE and MASSON staining to detect intestinal barrier damage 1. After euthanizing the mice by cervical dislocation, the abdomen was opened, the rectum was removed, the intestinal mucosa was separated, and the tissue was dehydrated in a fully enclosed tissue dehydrator and placed in xylene. After being embedded in a biological tissue embedding machine, the tissue was placed in a rotary microtome for routine pathological sectioning. After dewaxing, the tissue was hydrated and stained with hematoxylin and eosin (HE) using a hematoxylin and eosin (HE) staining kit (refer to the instruction manual for specific steps). The pathological morphology of the mouse intestinal mucosa was then observed under a microscope, and five images of the intestinal mucosa were randomly acquired.

[0044] Test results as follows Figure 2 As shown, from Figure 2 The results of HE staining showed that in the normal group, villi were dense and without shedding, crypt structure was normal, tissue showed no hemorrhage or ulceration, regular morphology, and no inflammatory cell infiltration. In the model group, the intestinal tissue showed incomplete intestinal epithelial structure, cell degeneration and shedding, microabscess formation in crypts, thinning of the intestinal mucosa, shortening of villi, capillary dilation, intestinal wall mucosal congestion and edema, and local or diffuse ulceration. Compared with the model group, after treatment, some intestinal epithelial cells regenerated, coagulative necrosis of villi and mucosa decreased, mucosal structure was more intact, inflammatory cell infiltration decreased, and the severity of lesions was significantly reduced. Furthermore, the degree of relief was dose-dependent, indicating that… L. plantarum CGMCC8198 can improve the pathological damage of intestinal mucosa tissue in mice with acute radiation enteritis.

[0045] 2. After euthanizing the mice by cervical dislocation, the abdomen was opened, the rectum was removed, the intestinal mucosa was separated, and the tissue was dehydrated in a fully enclosed tissue dehydrator and placed in xylene. After being embedded in a biological tissue embedding machine, the tissue was placed in a rotary microtome for routine pathological sectioning. After dewaxing, the tissue was hydrated and stained with Masson's trichrome staining kit (refer to the instruction manual for specific steps). The pathological morphology of the mouse intestinal mucosa was then observed under a microscope, and five random fields of view images were acquired.

[0046] Test results as follows Figure 2 As shown, from Figure 2 As can be seen from Masson's results, the intestinal barrier was severely damaged after radiation, but after gavage treatment, the intestinal mucosa remained relatively intact, and the degree of damage was dose-dependent, indicating that... L. plantarum CGMCC8198 can improve the pathological damage of intestinal mucosa tissue in mice with acute radiation enteritis.

[0047] Example 4: 16S rRNA Analysis Feces were collected from dissected mice for 16S rRNA analysis.

[0048] Test results as follows Figure 3 As shown, from Figure 3 As can be seen from the data, identification through 16S rRNA sequencing, and analysis using Alpha-species diversity analysis and the Observed species index revealed that, after... L. plantarum CGMCC8198 treatment improved radiation-induced reduction in gut microbiota abundance (value difference ≈ 70) and maintained gut microbiota stability. Heatmap analysis revealed differential gut microbiota among groups, and further analysis was conducted via gavage. L. plantarum The abundance of *Lactobacillus* genus in CGMCC8198 was higher than that in the model group. Enrichment analysis of the top 10 differentially expressed microbial communities revealed that... L. plantarum CGMCC8198 can protect beneficial bacteria in the gut, such as Eubacterium mucosae, and inhibit harmful bacteria, such as Helicobacter pylori.

[0049] Example 5: Western blot detection of PI3K and AKT factor expression Proteins were extracted using RTAEC assays prepared using a whole-cell lysis assay (KeyGen Bio Tech, Nanjing, China, KGP2100) and a dicaprylic acid (BCA) kit (KeyGen Bio Tech, KGP902). After protein concentration leveling, buffer was added and the samples were collected in centrifuge tubes for later use. Equal volumes of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [10% (w / v) gel dissolution] and electrotransferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked in 5% milk and washed with PBS. The membrane was then incubated overnight at 4°C with anti-PI3K and anti-AKT. After recovering the primary antibody and washing with PBS, the membrane was incubated with anti-rabbit rhizome peroxidase secondary antibody at 25°C for 2 hours. Final protein expression was detected by electrochemiluminescence using a Chemidoc imaging system (Bio-Rad, USA), and the optical density of each band was quantified using ImageJ software.

[0050] Test results as follows Figure 4 As shown, from Figure 4 As can be seen from the results, Western blot analysis of key factors in the PI3K-AKT-mTOR pathway revealed that the PI3K and AKT levels in the model group were significantly higher than those in the normal group. After treatment, the PI3K and AKT levels were significantly lower than those in the model group. The changes in the levels of each indicator after treatment were significant, and the decreasing trend was dose-dependent.

[0051] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. The application of a lactic acid bacterium via the PI3K-AKT-mTOR pathway in the preparation of a drug for treating acute radiation enteritis, wherein the lactic acid bacterium is *Lactobacillus plantarum* (…). Lactobacillus plantarum CGMCC No. 8198.

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