Application of phytobacterium plantarum GOLDGUT-HNU082 in relieving side effects of tumor drugs
By using the fungic agent of Lactobacillus Lactobacillus GOLDGUT-HNU082, liver and intestinal damage during gefitinib treatment was alleviated, and the problem of difficult to effectively alleviate drug side effects in the prior art was solved, and protection of liver and intestinal health and regulation of intestinal microecology was achieved.
Patent Information
- Application Number
- CN202510259055.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-06
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively alleviate the side effects of the non-small cell lung cancer targeted drug gefitinib, especially in the liver and intestines.
Using GOLDGUT-HNU082, through the preparation and application of its bacterial agent, the liver and intestinal damage caused by gefitinib is significantly reduced, and the intestinal microecology is regulated, and the production of short-chain fatty acids is improved.
It significantly reduced the level of liver aminotransferase and inflammatory markers during gefitinib treatment, protected the integrity of liver and intestinal tissue structure, and improved the production of intestinal short-chain fatty acids, enhancing intestinal health and immune function.
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Figure CN120093797A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and more specifically to application of Lactobacillus plantarum GOLDGUT-HNU082 in alleviating the side effects of tumor drugs. Background Art
[0002] The intestine is the largest immune organ in the human body, which controls more than 70% of the human immune function and produces about 80% of the resistance. As beneficial bacteria in the intestine, probiotics have the functions of regulating intestinal flora, promoting the absorption of nutrients, improving the body's immunity, inhibiting intestinal inflammation, and protecting the intestinal mucosal barrier. Although probiotics have many benefits, their interactions with drugs may also lead to some adverse effects. Probiotics may affect the individualized response of their hosts to drug treatment by degrading drugs. In some cases, the intake of probiotics may accelerate the chemical transformation of drugs, thereby affecting the role of drugs in specific locations. For example, studies have shown that probiotics may degrade the lipid-lowering drug lovastatin, but this effect does not necessarily affect the efficacy of the drug. Instead, it may play a beneficial role in slowing down the inflammatory response of the liver. However, this does not mean that all interactions between probiotics and all drugs are harmless.
[0003] In recent years, the incidence of lung cancer has shown an upward trend. Gefitinib is one of the targeted drugs for the treatment of lung cancer. It has good safety and tolerability. As an oral epidermal growth factor receptor tyrosine kinase inhibitor, it inhibits the activity of epidermal growth factor receptor tyrosine kinase to inhibit tumor growth, metastasis and angiogenesis, and increase tumor cell apoptosis. Gefitinib has a high oral bioavailability of 60% to 80%. It is rapidly absorbed in the gastrointestinal tract, mainly metabolized by the liver, and excreted through bile. Only a small part is excreted through urine in an unmetabolized form. However, gefitinib may also cause some adverse reactions of the digestive system, such as diarrhea and elevated transaminases. The presence of probiotics may have a positive effect on the therapeutic effect of gefitinib by alleviating its side effects, protecting intestinal health, and enhancing immune function, but there are no public reports yet.
[0004] Therefore, exploring probiotics that can alleviate the side effects of gefitinib is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention
[0005] In view of this, the present invention provides the use of Lactobacillus plantarum GOLDGUT-HNU082 in alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer.
[0006] In order to achieve the above object, the present invention adopts the following technical solution:
[0007] Application of Lactobacillus plantarum GOLDGUT-HNU082 in alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer. The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.
[0008] Another object of the present invention is to provide an application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a bacterial agent for alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer. The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.
[0009] Another object of the present invention is to provide a microbial agent, wherein the microbial agent comprises Lactobacillus plantarum GOLDGUT-HNU082 with a deposit number of CGMCC NO.33075.
[0010] Preferably, the microbial agent has the effect of alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer.
[0011] Another object of the present invention is to provide the use of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a drug for inhibiting non-small cell lung cancer cells, wherein the deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.
[0012] Beneficial effects: Gefitinib is a targeted therapeutic drug for EGFR gene mutation type non-small cell lung cancer, with clear efficacy, but its side effects limit its application. The plant lactobacillus GOLDGUT-HNU082 (deposit number CGMCC NO.33075) of the present invention can significantly reduce the liver and intestinal damage caused by gefitinib, which is manifested by reduced liver transaminase, reduced inflammatory markers, and more complete liver and intestinal tissue structure. In addition, the bacteria also significantly increased the production of short-chain fatty acids, further confirming its regulatory effect on intestinal microecology. This study aims to explore the mechanism of probiotics-mediated intestinal flora in alleviating drug side effects, promote probiotics to become a companion product of gefitinib drugs, reduce the psychological burden of patients taking medication and significantly improve drug side effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0014] Figure 1This is the colony morphology of Lactobacillus plantarum GOLDGUT-HNU082;
[0015] Figure 2 This is the whole genome comparison result of Lactobacillus plantarum GOLDGUT-HNU082.
[0016] Figure 3 is the degradation rate of gefitinib by different lactic acid bacteria.
[0017] Figure 4 The effect of Lactobacillus plantarum GOLDGUT-HNU082 metabolites on non-small cell lung cancer cells HCC827.
[0018] Figure 5 This is the effect of Lactobacillus plantarum GOLDGUT-HNU082 on tumor volume in lung cancer mice.
[0019] Figure 6 This is the effect of Lactobacillus plantarum GOLDGUT-HNU082 on tumor weight in lung cancer mice.
[0020] Figure 7 This is the effect of Lactobacillus plantarum GOLDGUT-HNU082 on liver function indicators in lung cancer mice.
[0021] Figure 8 The effect of Lactobacillus plantarum GOLDGUT-HNU082 on liver and intestinal tissues of lung cancer mice.
[0022] Fig. 9 This study is about the effect of Lactobacillus plantarum GOLDGUT-HNU082 on the content of short-chain fatty acids in the intestine of mice with lung cancer. DETAILED DESCRIPTION
[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0024] The embodiment of the present invention discloses the application of plant lactobacillus GOLDGUT-HNU082 in alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer, and the experimental drug gefitinib (pharmaceutical secondary standard) involved in the present invention is purchased from AstraZeneca UK Limited; the HCC827 cells involved are human non-small cell lung cancer cells, purchased from the cell resource platform constructed by Xiamen Yimo Biotechnology Co., Ltd.; the involved Lactobacillus paracasei H22B008, Lactobacillus paracasei H22B022, plant lactobacillus E22D048, salivary combined lactobacillus F22C005, and salivary combined lactobacillus F22E058 are all purchased from the tropical probiotic lactic acid bacteria resource library of Hainan University. The method involved in the present invention, if not specifically mentioned, is a conventional method, which will not be repeated here one by one.
[0025] All statistical analyses in the present invention were performed using "Graphpad Pism9.5" software for One-way ANOVA, and differences were considered significant based on a p-value threshold of 0.05 (*p<0.05, **p<0.01). Data are presented as mean ± standard error (SE). "Graphpad Pism9.5" was used for drawing.
[0026] Example 1
[0027] Isolation, Screening and Identification of Lactobacillus plantarum GOLDGUT-HNU082
[0028] 1. More than 30 fermented food samples were collected from farmers' markets in various counties and cities in Hainan Province, including Wanning, Wenchang, Wuzhishan, Baisha, and Lingshui. The collected fermented food samples were diluted tenfold in sterilized saline, and then a 10-fold dilution method was used to prepare a gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Sample diluent, 0.1mL of 10 -3 , 10 -4 , 10 -5 The gradient samples were spread on MRS agar medium and cultured anaerobically at 37°C for 48 hours. After multiple separations, streaking, purification, culture, and identification, 5 strains of Lactobacillus plantarum were screened out. The 5 strains were cultured in MRS liquid for 24 hours, and the OD value and viable count were measured. Finally, a strain of Lactobacillus plantarum was screened out and named GOLDGUT-HNU082. After the purified GOLDGUT-HNU082 strain was inoculated in MRS broth medium for activation for 24 hours, it was spread on MRS agar medium. After cultured at 37°C for 24 hours, the colonies were white, with a diameter of 0.1-0.2 cm, a smooth, moist and raised surface, and neat edges (such as Figure 1 Observed under an optical microscope, the bacteria are rod-shaped.
[0029] 2. Identification information: The whole genome of the GOLDGUT-HNU082 strain was sequenced. The sequence obtained by whole genome sequencing was used for genome alignment. The genome similarity analysis was performed using the online analysis platform Galaxy (https: / / usegalaxy.org / ), and the software used was FastANI (https: / / github.com / ParBLiSS / FastANI). The analysis results showed that the ANI value of the whole genome sequence of GOLDGUT-HNU082 and Lactiplantibacillus plantarum SRCM100442 was 99.0642 (e.g. Figure 2 shown).
[0030] 3. The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO.33075, the deposit date is December 16, 2024, and the taxonomic name is Lactiplantibacillus plantarum.
[0031] Example 2
[0032] Effect of Lactobacillus plantarum GOLDGUT-HNU082 on gefitinib
[0033] 1. Degradation rate of gefitinib
[0034] According to previous studies, the concentration of gefitinib mother solution was determined to be 33 μM. The culture medium: bacterial solution: gefitinib mother solution = 12 mL: 400 μL: 200 μL were mixed evenly, and the mixture was co-cultured for 0 hours, 6 hours, 12 hours and 24 hours. The expression peak area was measured using an Agilent Technologies high performance liquid chromatograph (chromatographic column: octadecylsilane bonded silica gel column; column temperature: 30 °C; mobile phase: ammonium acetate solution: acetonitrile = 65:35; flow rate: 1.0 mL / min detection: 243 nm ultraviolet detection). Gefitinib cultured in the culture medium alone was used as a blank control. The peak area of gefitinib cultured in conjunction with probiotics was compared with the peak area of gefitinib to calculate the degradation rate of gefitinib by probiotics.
[0035] The different types of probiotics are Lactobacillus plantarum GOLDGUT-HNU082, Lactobacillus paracasei H22B008, Lactobacillus paracasei H22B022, Lactobacillus plantarum E22D048, Lactobacillus salivarius F22C005, and Lactobacillus salivarius F22E058.
[0036] The calculation formula of gefitinib degradation rate is as follows:
[0037] V=(AB) / A
[0038] V: Degradation rate of gefitinib by probiotics
[0039] A: Peak area of gefitinib
[0040] B: Peak area of gefitinib after synergistic probiotic culture
[0041] The results are attached Figure 3 As shown in the figure, among different probiotics, Lactobacillus plantarum GOLDGUT-HNU082 had the lowest degradation rate of gefitinib.
[0042] 2. Effects on non-small cell lung cancer cells
[0043] HCC827 cells were grown in DMEM medium supplemented with 10% fetal bovine serum and antibiotics (1% penicillin + 1% streptomycin). The cells were placed in a fully humidified, 5% CO atmosphere at 37°C. 2 All experiments were performed on cells in the logarithmic growth phase. The medium was changed every 2 days and the cells were subcultured using 0.25% phenol red-free trypsin.
[0044] After trypsinization and collection of cells, 1 ml of culture medium was added and mixed to prepare a suspension. The cells were then counted and adjusted to approximately 2,000 cells per ml.
[0045] After cell counting, prepare a 96-well culture plate. Subsequently, HCC827 cells were evenly distributed into each well of the plate, and the culture plate was placed in a 37°C incubator. After incubation for 48 hours, during which time the cells grew normally and were confluent, the probiotics were inoculated on the MRS broth medium, and the probiotic culture reached the maximum growth period by centrifugation to obtain the supernatant at a speed of 6000 rpm for 3 minutes, and then the supernatant was removed on a clean bench, sterilized with a 0.22 μM microporous filter membrane, and stored at -80°C. The metabolite supernatant of Lactobacillus plantarum GOLDGUT-HNU082 was used as a diluent with cell culture medium, and the concentrations of 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM Lactobacillus plantarum GOLDGUT-HNU082 metabolite solutions were accurately prepared in sequence according to different concentrations. After preparation, according to the experimental design, the metabolite solutions of each concentration were accurately added to the corresponding cell culture wells to ensure the accuracy and consistency of the added amount, so as to meet the requirements of different concentration conditions for subsequent cell experiments. The culture plate was then incubated for another 24 hours, and the cell viability was assessed using the MTT assay. After 24 hours of cell treatment, the culture medium was carefully removed and 100 μL of MTT dye solution was added to each well. The plate was then placed in the incubator for another 4 to 6 hours. After incubation, 150 μL of DMSO was added to each well and shaken for 10 minutes to fully dissolve the crystals. Subsequently, a microplate reader (Tecan 200PRO model, Switzerland) was used to detect the absorbance (OD) at a wavelength of 570 nm.
[0046] The results are attached Figure 4 As shown, when coexisting with 20 μM of Lactobacillus plantarum GOLDGUT-HNU082 metabolites, the survival rate of lung cancer cells quickly reached the lowest point. This result suggests that Lactobacillus plantarum GOLDGUT-HNU082 has a significant inhibitory effect on HCC827 cells.
[0047] 3. Effects on lung cancer mice
[0048] (1) Methods:
[0049] This study was approved by the Animal Ethics Committee of Hainan University, and all animal operations were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hainan University. 24 female SPF-grade BALB / c Nude mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (4 weeks old). The nude mice were housed under standard laboratory conditions of day / night: 12 / 12 hours, humidity: 60%, and temperature: 23°C, with unlimited access to standard food diet and drinking water. The nude mice were allowed to acclimate to the environment for one week before the experiment. Both the control group and the lung cancer model group consumed a maintenance basal feed, which was provided by Beijing Keao Xieli Feed Co., Ltd.
[0050] The HCC827 cells were diluted with 1×PBS:Matrigel = 1:1 to adjust the concentration to 1×10 per ml 7 The cell suspension was placed on ice and then quickly injected subcutaneously near the left hind leg of the nude mouse. The injection volume of each mouse was 200 μL. After inoculation of HCC827 cells, an obvious mass was touched at the inoculation site 14 days later, indicating that the tumor cells had successfully proliferated and formed tumors in the nude mice, and the model was successfully established. The mice with successful modeling were treated with gefitinib, and the appropriate dosage and course of treatment were set. The changes in tumor volume and weight change of the mice were observed and recorded. If the tumor growth of the mice was significantly inhibited after gefitinib treatment, the tumor volume was reduced or the growth rate was significantly slowed down, and the weight of the mice remained relatively stable or had no obvious adverse reactions, it indicated that the modeling mice had a good therapeutic response to gefitinib and met the indications of gefitinib. The weight and tumor volume of the mice were recorded regularly thereafter. The tumor volume was calculated by (L×W×H) / 2, where L is the tumor length, W is the tumor width, and H is the height.
[0051] When the tumor volume of nude mice is greater than 50 mm 3 , and they were grouped according to their tumor volume. There were 4 groups (n=6) in this study, namely: blank control group (Control, CON); non-small cell lung cancer model group (Model, MO); gefitinib-treated non-small cell lung cancer model group (Model+Gefitinib, Gef); gefitinib synergistically with Lactobacillus plantarum GOLDGUT-HNU082 treated non-small cell lung cancer model group (Model+Gefitinib+Lactobacillus plantarum GOLDGUT-HNU082, G+HNU082).
[0052] After the grouping was completed, the nude mice were treated for 6 weeks. The CON and MO groups were gavaged with 200 μL of 0.5% sodium carboxymethyl cellulose solution every day; the Gef group was gavaged with 200 μL of 100 mg / KG gefitinib (dissolved in 0.5% sodium carboxymethyl cellulose) every day; the G+HNU082 group was gavaged with 200 μL of 100 mg / KG gefitinib + 10 9 cfu Lactobacillus plantarum GOLDGUT-HNU082 (dissolved in 0.5% sodium carboxymethylcellulose). The body weight was weighed once a week and the tumor volume was measured once a week.
[0053] After 6 weeks of treatment, the effect of the presence of probiotics on the therapeutic effect of gefitinib was determined.
[0054] The feces of the mice were collected once before and after the treatment, and stored at -80°C in time for metagenomic determination. After 6 weeks of treatment, blood, tissue samples and colon contents of the mice were collected under sterile conditions. The blood was centrifuged to obtain serum, and the serum and colon contents were stored at -80°C until use, for the determination of transaminases and inflammatory factors in nude mice. The serum was used to determine indicators such as transaminases, bilirubin, and creatinine, and the colon contents were used to determine short-chain fatty acids. The dissected liver was divided into two parts, one was placed in a sterile centrifuge tube for quick freezing in liquid nitrogen and then frozen at -80°C, and one was rinsed with 0.85% saline and fixed in a paraformaldehyde solution for liver tissue sectioning (Wuhan Savier Biotechnology Co., Ltd.); the dissected small intestine was placed in paraformaldehyde for fixation to make intestinal sections (Wuhan Savier Biotechnology Co., Ltd.). Short-chain fatty acids were determined using gas chromatography-mass spectrometry analysis by Agilent Technologies Inc. (column Agilent DB-WAX: 0.25 mm × 0.25 μm × 50 cm; inlet temperature: 250°C; gas interface temperature: 250°C; carrier gas flow rate: 1.5 mL / min; split ratio = 3:1; split ratio: 1 μL).
[0055] (2) Results:
[0056] Tumor volume and weight: Results are as attached Figure 5 and attached Figure 6 As shown, after 6 weeks of treatment, there was no significant difference in tumor volume and tumor weight between the gefitinib group (Gef) and the gefitinib combined with Lactobacillus plantarum GOLDGUT-HNU082 group (G+HNU082), indicating that when gefitinib is used to treat lung cancer, the simultaneous intake of Lactobacillus plantarum GOLDGUT-HNU082 does not affect its drug treatment effect.
[0057] Liver function index: Results as attached Figure 7 As shown in the figure, compared with the model group, the serum index levels of the gefitinib group, including alanine aminotransferase, glutamate aminotransferase, and blood creatinine, were significantly increased (p<0.01), and total bilirubin was also significantly increased (p<0.05). However, compared with the gefitinib group, the gefitinib-co-Lactobacillus plantarum GOLDGUT-HNU082 group was able to significantly reduce the serum alanine aminotransferase, glutamate aminotransferase, total bilirubin, and blood creatinine (p<0.01). This indicates that Lactobacillus plantarum GOLDGUT-HNU082 significantly alleviated the liver damage caused by gefitinib.
[0058] Liver and intestine tissue sections: as attached Figure 8As shown in the results of liver tissue sections, the hepatocyte ballooning degeneration in the gefitinib-cooperated Lactobacillus plantarum GOLDGUT-HNU082 group was less than that in the gefitinib group; the lymphocyte infiltration in the intestinal tissue sections in the gefitinib-cooperated Lactobacillus plantarum GOLDGUT-HNU082 group was significantly less than that in the gefitinib group. It can be seen that when gefitinib is used to treat non-small cell lung cancer, it will cause inflammatory reactions in the liver and intestines of nude mice, and when it is taken in combination with the probiotic Lactobacillus plantarum GOLDGUT-HNU082, the inflammatory reaction can be effectively alleviated.
[0059] Intestinal short-chain fatty acid content: as shown in the attached Fig. 9 As shown in the figure, the contents of acetate, isovaleric acid, and caproic acid in the gefitinib-assisted Lactobacillus plantarum GOLDGUT-HNU082 group were significantly higher than those in the gefitinib group (p<0.05), and the contents of propionate, butyrate, and isobutyrate in the gefitinib-assisted Lactobacillus plantarum GOLDGUT-HNU082 group were extremely significantly higher than those in the gefitinib group (p<0.01). This suggests that when we use gefitinib to treat lung cancer, supplementing Lactobacillus plantarum GOLDGUT-HNU082 can increase the content of short-chain fatty acids in the body to a certain extent. As a product of intestinal microbial fermentation of indigestible carbohydrates, short-chain fatty acids play a key role in maintaining intestinal health. Physiologically, it accurately regulates the proliferation and differentiation of intestinal epithelial cells, maintains the intestinal barrier, and resists pathogens; at the immune level, it affects the activity of immune cells and the secretion of cytokines, enhances immunity, and reduces inflammation, such as butyrate inhibits the expression of inflammatory factors. At the same time, short-chain fatty acids regulate the intestinal microbial community, create a suitable environment for beneficial bacteria, inhibit harmful bacteria, and reduce harmful metabolites. For example, propionic acid protects intestinal health and also participates in the regulation of the intestinal nervous system to maintain normal digestive function.
[0060] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0061] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Application of Lactobacillus plantarum GOLDGUT-HNU082 in alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.
2. Application of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a bacterial agent for alleviating the side effects of the non-small cell lung cancer targeted drug gefitinib, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCCNO.33075.
3. A microbial agent, characterized in that: The microbial agent includes Lactobacillus plantarum GOLDGUT-HNU082 with a preservation number of CGMCC NO.33075.
4. The microbial agent according to claim 3, characterized in that: The microbial agent has the effect of alleviating the side effects of gefitinib, a targeted drug for non-small cell lung cancer.
5. Use of Lactobacillus plantarum GOLDGUT-HNU082 in the preparation of a drug for inhibiting non-small cell lung cancer cells, characterized in that: The deposit number of the Lactobacillus plantarum GOLDGUT-HNU082 is CGMCC NO.33075.