Application of lactobacillus reuteri in preparation of medicine for preventing and treating blastozoon infection

Abstract

The invention discloses application of lactobacillus reuteri in preparation of a medicine for preventing and treating cystic protozoa infection. The method comprises the following steps: collecting a machin excrement sample, carrying out cystoprotozoa identification, selecting a cystoprotozoa positive sample, and determining the absolute number of cystoprotozoa in the cystoprotozoa positive sample; then metagenome sequencing analysis shows that the relative abundance of the lactobacillus reuteri is in significant negative correlation with the absolute number of the cystoprotozoa, which indicates that the lactobacillus reuteri can inhibit the growth and reproduction of the cystoprotozoa to a certain extent; finally, a treatment experiment of the in-vivo lactobacillus reuteri on the cystoprotozoa is carried out by establishing a machin animal model, and a result shows that the number of the cystoprotozoa in the machin after treatment is obviously reduced compared with that before treatment, so that the lactobacillus reuteri A21041 strain can be used for effectively treating cystoprotozoa infection; the compound has a good application prospect in preparation of medicines for preventing and treating cystozoon infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of Lactobacillus reuteri, and particularly to the application of Lactobacillus reuteri in the preparation of medicines for preventing and treating Blastocystis infection. Background Art

[0002] Blastocystis is a common parasite that is widely found in the intestines of humans and a variety of animals. It is mainly transmitted through the fecal-oral route and can cause clinical symptoms such as diarrhea, abdominal pain and vomiting. Especially for immunocompromised patients (such as children, the elderly and AIDS patients), the risk of Blastocystis infection is higher. Blastocystis infection not only seriously threatens the health of patients, but also imposes a huge burden on the public health system.

[0003] At present, the treatment options for Blastocystis infection are relatively limited, mainly relying on drug therapy, such as antiprotozoan drugs such as metronidazole and tinidazole. However, these drugs are often accompanied by significant side effects during treatment, including gastrointestinal discomfort and allergic reactions, and some drugs may develop drug resistance during long-term use. In addition, the therapeutic effect of traditional drugs is not ideal, and it cannot effectively prevent repeated infection of Blastocystis. Therefore, finding an effective, safe, and side-effect-free therapeutic drug has become an important issue that needs to be solved urgently.

[0004] Lactobacillus reuteri is a probiotic widely found in the intestines of humans and animals. Its effects are mainly based on regulating the balance of intestinal flora, inhibiting the growth of pathogens, and regulating the immune system. At present, there are no reports on the use of Lactobacillus reuteri in the prevention and treatment of Blastocystis infection. Summary of the invention

[0005] The present invention aims to provide the use of Lactobacillus reuteri in the preparation of a medicament for preventing and treating Blastocystis infection, so as to effectively inhibit the growth and reproduction of Blastocystis, alleviate the clinical symptoms caused by Blastocystis, and reduce the frequency of use of antibiotics.

[0006] The invention discloses an application of Lactobacillus reuteri in preparing a medicine for preventing and treating Blastocystis infection. The Lactobacillus reuteri is strain A21041.

[0007] As a further preference of the technical solution of the present invention, the drug can be used by both humans and animals. Calculated based on the human body weight, the effective amount is 0.02 - 0.04 g / kg (referring to the dosage in the patent "A Lactobacillus reuteri strain for prolonging lifespan, anti-aging, and reducing fat, and its products and applications"); the effective dosage for animals is calculated according to the conversion formula in "Dose Conversion Between Animals and Humans: A Practical Solution", specifically: animal dosage = human dosage × (animal body weight / human body weight). 0.33 The applicable animals include but are not limited to mice, rabbits, dogs, pigs, etc.

[0008] Furthermore, the drug type is any pharmaceutically acceptable oral fungal drug dosage form; preferably, it is freeze-dried powder of Lactobacillus reuteri, bacterial liquid, or granular inoculum. The administration frequency is once every 1 - 24 h.

[0009] The present invention also provides a pharmaceutical composition for preventing and treating Blastocystis infection, and the pharmaceutical composition contains the above-mentioned Lactobacillus reuteri.

[0010] The beneficial effects of the present invention are as follows:

[0011] In the present invention, fecal samples of cynomolgus monkeys are collected for Blastocystis identification, and positive samples of Blastocystis are selected for the determination of the absolute number of Blastocystis therein; then through metagenomic sequencing analysis, it shows that the relative abundance of Lactobacillus reuteri is significantly negatively correlated with the absolute number of Blastocystis, indicating that Lactobacillus reuteri can inhibit the growth and reproduction of Blastocystis to a certain extent; finally, by establishing a cynomolgus monkey animal model and conducting in vivo treatment experiments of Lactobacillus reuteri on Blastocystis, the results show that the number of Blastocystis in cynomolgus monkeys after treatment is significantly reduced compared with that before treatment, indicating that Lactobacillus reuteri strain A21041 can effectively treat Blastocystis infection and has good application prospects in the preparation of drugs for preventing and treating Blastocystis infection. Description of the Drawings

[0012] Figure 1 It is an electrophoresis diagram for PCR identification of Blastocystis;

[0013] Figure 2 It is a diagram of Sanger sequencing identification results;

[0014] Figure 3 It is the correlation between the relative abundance of Lactobacillus reuteri and the absolute number of Blastocystis;

[0015] Figure 4 It is a design diagram for treating cynomolgus monkeys with Lactobacillus reuteri;

[0016] Figure 5Change in the absolute number of Blastocystis in fecal samples of cynomolgus monkeys before and after treatment with Lactobacillus reuteri strain A21041. Detailed implementation mode

[0017] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0018] Example 1: Collection of cynomolgus monkey fecal samples and identification of Blastocystis

[0019] 98 fecal samples of cynomolgus monkeys were collected from the monkey farm, DNA was extracted using a fecal DNA extraction kit, and qualitative and quantitative identification of Blastocystis in the fecal samples was performed by combining PCR and qPCR methods.

[0020] (1) Qualitative detection: PCR amplification solution (2×Taq PCR Mix, Takara) and amplification primers were added to the extracted DNA for PCR amplification (the amplification system is shown in Table 1).

[0021] Table 1 PCR amplification system for Blastocystis

[0022]

[0023]

[0024] The PCR amplification primers for Blastocystis refer to the literature (Scicluna SM, Tawari B, Clark CG. DNA barcoding of blastocystis. Protist. 2006;157(1):77 - 85.). The primer sequences are as follows:

[0025] Forward primer BhRDr: 5′-GAGCTTTTTAACTGCAACAACG-3′

[0026] Reverse primer RD5: 5′-ATCTGGTTGATCCTGCCAGT-3′

[0027] The amplification program was: 95°C for 3 min; 94°C for 1 min, 65°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 2 min. The amplification products were identified by gel electrophoresis and Sanger sequencing.

[0028] Qualitative detection results: The gel electrophoresis results of the PCR products are as Figure 1 shown, and the amplified fragment size is 600 bp; the Sanger sequencing identification results are as Figure 2 shown;

[0029] From Figure 1 and Figure 2As can be seen from the results, among the 98 fecal samples of cynomolgus monkeys collected from Houchang, 96 were positive for Blastocystis, with an infection rate of 97.9%.

[0030] (2) Statistics of the absolute number of Blastocystis in positive cynomolgus monkey fecal samples (quantitative detection): For Blastocystis-positive samples, quantitative detection was performed by qPCR. Specifically: qPCR amplification solution (2× qPCR Master Mix, Promega) and amplification primers were added to the DNA of positive samples for qPCR amplification (the amplification system is shown in Table 2). A qPCR amplification standard curve was established using the genomic DNA of in vitro-cultured Blastocystis. The CT value of the sample was compared with the CT value of the standard curve, and the absolute number of Blastocystis per milligram of feces was calculated.

[0031] Table 2 Blastocystis qPCR amplification system

[0032]

[0033] The qPCR amplification primers for Blastocystis refer to the literature (Poirier P, Wawrzyniak I, Albert A, et al. Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. J Clin Microbiol. 2011;49(3):975 - 983.). The primer sequences are as follows:

[0034] Forward primer BL18SPPF1: 5′-AGTAGTCATACGCTCGTCTCAAA-3′

[0035] Reverse primer BL18SR2PP: 5′-TCTTCGTTACCCGTTACTGC-3′

[0036] The amplification program was: 95℃ for 2 min; 95℃ for 15 s, 68℃ for 1 min, for 45 cycles.

[0037] The quantitative detection results are shown in Table 3.

[0038] Table 3 The absolute number of Blastocystis in each sample

[0039] ID Absolute quantity ID Absolute quantity ID Absolute quantity ID Absolute quantity SX1 5.87260103 SX27 3.22708672 SX54 619.818689 SX80 36.2976468 SX2 24.7088039 SX29 9.11703535 SX55 40.8797438 SX81 236.071784 SX3 67.2904936 SX30 0.1426395 SX56 42.0568914 SX82 7.8577824 SX4 15.4049442 SX31 1.16370177 SX57 10.2843949 SX83 12.8697334 SX5 54.4854409 SX32 1.05090953 SX58 16.2172561 SX84 15735.8841 SX6 8.88307301 SX33 0.43354014 SX59 64.0302982 SX85 171.625382 SX7 56.1482362 SX35 1.44403307 SX60 5.03340166 SX86 365.450522 SX8 14.9164895 SX36 12.1157444 SX61 1.98964496 SX87 341.738647 SX9 22.2811569 SX37 1.69390703 SX62 18.7771202 SX88 1.01724106 SX10 52.7629495 SX39 0.14549976 SX64 22.6673442 SX89 24.2056952 SX12 13.9896352 SX40 17.5759153 SX65 12.5248765 SX90 45.6420924 SX13 46.2690782 SX41 43.9755173 SX66 5996.60868 SX91 30.8116783 SX14 2.18674354 SX42 143.266417 SX67 0.442017 SX92 825.458777 SX15 0.73539943 SX43 83.6643141 SX68 3.60035199 SX93 4166.73441 SX16 10.1568281 SX44 31.201499 SX69 7.04538065 SX94 175.735297 SX17 1.13325959 SX45 84.7152812 SX70 6.16740869 SX95 46.0588229 SX18 14.2446398 SX46 8.9278362 SX71 0.48445467 SX96 164.258875 SX19 58.1737663 SX47 15.556025 SX73 110.838464 SX97 39.3348383 SX20 6.96541269 SX48 12.3226646 SX74 0.47035171 SX98 38.4733989 SX22 0.71706666 SX49 65.3235681 SX75 45.1089677 SX99 262.053789 SX23 11.7331657 SX50 219.182878 SX76 0.56312585 SX101 360.668608 SX24 0.2320825 SX51 1.69399036 SX77 26.3113321 SX102 17.995277 SX25 7.56848324 SX52 18.6156367 SX78 9.73199421 SX103 28.6538148 SX26 1.27966646 SX53 1.98288002 SX79 1.76139026 SX104 11.913144

[0040] Example 2: Metagenomic sequencing analysis

[0041] For the Blastocystis-positive samples (n = 96), metagenomic sequencing was further performed. According to the metagenomic sequencing data, the correlation between the absolute number of Blastocystis in each sample and the gut microbiota was analyzed.

[0042] The results are as Figure 3 shown Figure 3 The results showed that the relative abundance of Lactobacillus reuteri was significantly negatively correlated with the absolute number of Blastocystis, indicating that Lactobacillus reuteri can inhibit the growth and reproduction of Blastocystis to a certain extent.

[0043] Example 3: In vivo treatment experiment of Lactobacillus reuteri on Blastocystis

[0044] To further prove that Lactobacillus reuteri can effectively inhibit the growth and reproduction of Blastocystis, the applicant conducted an in vivo experiment using a cynomolgus monkey animal model. The specific implementation method is as follows:

[0045] (1) Strain selection: Lactobacillus reuteri strain A21041 used in the present invention is derived from the gut microbiota sample of a centenarian. This strain is provided by Guangxi Aisheng Life Science Co., Ltd., China (Patent No.: CN115948292A) and is deposited in the Guangdong Microbial Culture Collection Center (GDMC), with the deposit number GDMCC No: 62832. First, viable cell counting was performed. At the time of factory shipment, the viable cell content of the freeze-dried powder was 1.54×10 11 CFU / g. Carefully open the freeze-dried powder bag in a safety cabinet, weigh 0.077 g of the powder, dissolve it with sterile PBS and then perform serial dilution (0, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 and 10 6 ). Then, 50 μL of the bacterial suspension was pipetted and inoculated onto MRS solid medium. Three replicates were made for each dilution. After culturing at 37 °C under anaerobic conditions for 24 h, viable cell counting was performed. The final measured viable cell content was 6.74×10 10 CFU / g.

[0046] (2) Treatment plan: A total of 11 cynomolgus monkeys infected with Blastocystis were included in this treatment (as Figure 4as shown). The freeze-dried powder was divided into 2 g / tube and stored in the dark. During the experiment, 2 g of the freeze-dried powder was dissolved in 130 mL of physiological saline and fed on an empty stomach. The daily feeding dose for each cynomolgus monkey was 1×10 10 CFU. The treatment lasted for 21 days. Fecal samples were collected 24 h before treatment and 24 h after treatment ended, and fecal DNA was extracted. The qPCR method was used to evaluate the number of Blastocystis in each milligram of feces before and after treatment.

[0047] (3) Treatment results: The results of this experiment are shown in detail in Figure 5 . To evaluate the effect of Lactobacillus reuteri strain A21041 on treating Blastocystis, the applicant conducted a quantitative detection of Blastocystis in the fecal samples of cynomolgus monkeys before treatment (day 0) and after treatment (day 21). The results of statistical analysis showed that the number of Blastocystis in cynomolgus monkeys decreased significantly after treatment compared with before treatment. Thus, it was indicated that Lactobacillus reuteri strain A21041 could effectively treat Blastocystis infection.

[0048] In summary, the present invention provides a method for preventing and treating Blastocystis infection using Lactobacillus reuteri. Through cohort studies and in-vivo experiments on animal models, it was verified that Lactobacillus reuteri has significant preventive and therapeutic effects, and is highly safe and has no side effects. Therefore, the present invention has good application prospects and can provide a new treatment strategy for Blastocystis infection.

Claims

1. Application of Lactobacillus reuteri in the preparation of medicines for preventing and treating Blastocystis infection.

2. The use of Lactobacillus reuteri as claimed in claim 1 in the preparation of a medicament for preventing and treating Blastocystis infection, characterized in that: The Lactobacillus reuteri is strain A21041.

3. Use of Lactobacillus reuteri as claimed in claim 1 or 2 in the preparation of a medicament for preventing and treating Blastocystis infection, characterized in that: The drug type is any pharmaceutically acceptable oral fungus drug dosage form.

4. The use of Lactobacillus reuteri as claimed in claim 3 in the preparation of a medicament for preventing and treating Blastocystis infection, characterized in that: The drug dosage form is Lactobacillus reuteri freeze-dried powder, bacterial liquid or granular inoculated bacterial agent.

5. A pharmaceutical composition for preventing and treating Blastocystis infection, characterized in that: The pharmaceutical composition comprises the Lactobacillus reuteri according to any one of claims 1, 2, and 4.

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