Application of metabolism-inactivated bacteria in relieving drug toxicity caused by nano-plastics

Abstract

The invention discloses an application of metabolic inactivated bacteria in relieving drug toxicity caused by nano-plastics, and belongs to the technical field of microorganisms. The caenorhabditis elegans is exposed to a polystyrene nano-plastic group, a medicine group and a nano-plastic and medicine combined exposure group respectively, and is fed with different foods, namely normal metabolism E.coli OP50 bacteria and metabolism inactivation E.coli OP50 bacteria respectively. It is found that the exercise behavior ability and the eating ability of caenorhabditis elegans fed with E.coli OP50 subjected to combined exposure of polystyrene nano-plastics and drugs and subjected to metabolism inactivation treatment are remarkably improved, and development delay is remarkably relieved. It is indicated that bacterial metabolism inactivation can relieve the toxicological effect of polystyrene nano-plastic and medicine combined exposure on caenorhabditis elegans. According to the invention, caenorhabditis elegans is taken as a model organism, the test period is short, the result is accurate, and technical support is provided for research of drug toxicity caused by bacterial metabolism participating in environmental pollutants.

Description

Technical Field

[0001] The invention relates to application of metabolically inactivated bacteria in alleviating drug toxicity caused by nano plastics, and belongs to the technical field of microorganisms. Background Art

[0002] The possible adverse effects of nanoplastics have recently received great attention. Nanoplastics are characterized by small size, large specific surface area, good hydrophobicity, and difficulty in degradation. Therefore, they are not only prone to accumulation in the tissues of multiple organs, but also easy to pass through cell membranes, enter cells, and act as carriers of harmful substances and microorganisms. Nanoplastics do not exist in isolation in the natural environment; they exhibit small particle size, wide specific surface area, and obvious hydrophobic properties. This enhances their ability to adsorb environmental pollutants, including heavy metals, as well as persistent organic and antibiotic pollutants. In addition, nanoplastics interact with these coexisting toxic substances and produce comprehensive toxic effects on organisms. Studies have shown that the gastrointestinal tract is the main site of nanoplastic accumulation and can cause serious effects on the digestive system. Nanoplastics can also enter the blood, lymphatic system and liver. Nanoplastics in the intestine may also affect the immune response of the digestive system. In addition to strict control of nanoplastics in environmental governance, we should also pay close attention to the impact of intestinal microorganisms on the toxicity of nanoplastics and their possible harm to human health.

[0003] As the main diet of C. elegans in the laboratory, Escherichia coli OP50 may participate in drug metabolism through its own activity, affecting the nutrients available to nematodes and the effects of drugs. For example, exposure to 5-fluoro-2'-deoxyuracil (FUdR) changes the differential expression of many OP50 genes, including genes for amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. The anti-diabetic drug metformin further extends the lifespan of C. elegans by disrupting the bacterial folate and methionine cycles. To eliminate the metabolic effects of live bacteria, we used a method of inactivating bacteria here. The treated bacteria have no growth or metabolic activity, but are still an edible and nutritious food source for C. elegans and can be used throughout the life cycle of C. elegans. Currently, no articles provide the application of metabolically inactivated bacteria in alleviating drug toxicity caused by nanoplastics. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the application of metabolically inactivated bacteria in alleviating drug toxicity caused by nanoplastics. The present invention develops a rapid and low-cost method for inactivating bacteria. The bacteria treated by this method have no growth or metabolic activity, but are still an edible and nutritious food source for C. elegans, which can be used throughout the life cycle of C. elegans. And by feeding different foods: normal metabolic E. coli OP50 bacteria and metabolically inactivated E. coli OP50 bacteria, the role of metabolically inactivated bacteria in alleviating tamoxifen toxicity caused by nanoplastics was further evaluated.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The method comprises the following steps: adding different foods into NGM solid culture medium: normal metabolic E. coli OP50 bacteria and metabolically inactivated E. coli OP50 bacteria, and then inoculating L1 stage Caenorhabditis elegans into NGM solid culture medium containing different foods including polystyrene nanoplastics (PS-NPs), tamoxifen, and a mixture of polystyrene nanoplastics and tamoxifen, and culturing for 3 days, and evaluating the effect of metabolically inactivated bacteria in alleviating the tamoxifen toxicity caused by nanoplastics based on the head swing frequency, body movement frequency, pharyngeal pumping frequency, reproductive rate and development of the Caenorhabditis elegans.

[0007] In the present invention, a lysis solution is used to lyse Caenorhabditis elegans in the egg-laying period to obtain sufficient eggs. After culturing for 18 to 22 hours, the nematodes hatched to the L1 stage are transferred to a blank control group, a single exposure experimental group, and a combined exposure experimental group containing different foods. After the exposure period, the effect of metabolically inactivated bacteria in alleviating the tamoxifen toxicity caused by nanoplastics is evaluated based on at least one of the head swing frequency, body movement frequency, pharyngeal pumping frequency, reproductive rate and area of ​​the Caenorhabditis elegans.

[0008] Under the combined exposure conditions of nanoplastics and tamoxifen, when the measured indicators of the metabolically inactivated bacteria group were significantly different from those of the normal metabolic bacteria group (P<0.05), the metabolically inactivated bacteria had an impact on the toxic effects of nanoplastics and tamoxifen.

[0009] According to the experiment, under the conditions of metabolically inactivated bacteria treatment, the toxicity-intensifying effect of low-concentration tamoxifen (0.1-10μM) caused by the concentration range of 0.1-10ug / L nanoplastics can be alleviated.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention found that the movement behavior ability and feeding ability of E. coli OP50 treated with combined exposure to polystyrene nanoplastics and tamoxifen and feeding metabolism inactivation were significantly improved, and the developmental delay was significantly alleviated. This shows that bacterial metabolic inactivation can alleviate the toxicological effects of combined exposure to polystyrene nanoplastics and tamoxifen on Caenorhabditis elegans. The present invention uses Caenorhabditis elegans as a model organism, has a simple method, low cost, short test cycle, and accurate results, and provides technical support for the study of drug toxicity caused by bacterial metabolism participating in environmental pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the toxic effect of the metabolically inactivated bacteria in the present invention on the body bending of nematodes under the combined exposure of polystyrene nanoplastics and tamoxifen.

[0013] Figure 2 This is the toxic effect of the metabolically inactivated bacteria in the present invention on the head swing of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen.

[0014] Figure 3 This is the toxic effect of the metabolically inactivated bacteria in the present invention on the pharyngeal pumping frequency of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen.

[0015] Figure 4 This is the toxic effect of the metabolically inactivated bacteria in the present invention on the reproduction of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen.

[0016] Figure 5 This is the toxic effect of the metabolically inactivated bacteria in the present invention on the development of nematodes under combined exposure to polystyrene nanoplastics and tamoxifen. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0018] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0019] Cultivation of Caenorhabditis elegans: Cenorhabditis elegans is cultured and propagated using nematode growth medium (NGM). A layer of OP50 (Escherichia coli strain OP50) colonies is coated on the surface of the medium and cultured in a constant temperature incubator at 20°C. NGM solid medium: 3g sodium chloride (NaCl), 2.5g peptone and 17g agar are weighed and added to 1L with double distilled water; after sterilization at 121°C for 20min, cool to 55°C, add 1mL 1M CaCl2, 1mL 1M MgSO4, 1mL 5M cholesterol solution, and 25mL 1M KHPO4 buffer, mix well, and pour into a culture dish.

[0020] Synchronization of Caenorhabditis elegans: Rinse the NGM culture plate with egg-laying nematodes repeatedly with K-buffer buffer, collect the egg-laying nematodes in the EP tube, wash the nematodes repeatedly with K solution, and discard the supernatant. Add the lysis solution to the EP tube, shake and lyse repeatedly to obtain enough eggs, and collect the nematodes hatched to the L1 stage with K solution after 18-22 hours of culture, centrifuge and discard the supernatant, add K solution, and inoculate into NGM culture medium for culture. K solution: 1.4902g sodium chloride, 1.1928g potassium chloride, 0.4g sodium acetate, use ddH2O to make up to 500mL, sterilize at high temperature and high pressure, and store at room temperature. 1M KHPO4 buffer (pH = 6.0): weigh 10.83g KH2PO4, 4.66g K2HPO4·3H2O, add double distilled water to 100mL, shake until the particles are dissolved, put into an autoclave at 121℃ for 20min, wait for the solution to cool to room temperature, and store in a 4℃ refrigerator.

[0021] Preparation of normal metabolic OP50: Two bottles of LB liquid culture medium sterilized at high temperature and high pressure, one bottle as blank control, add 100μL of E. coli OP50 bacterial solution to one bottle, and culture on a shaker at 37℃ and 170rpm for 4h until the culture medium becomes turbid. If the blank LB culture medium is clear, it can be placed at 4℃ for standby use. LB liquid culture medium: weigh 10g tryptone, 5g sodium chloride (NaCl), 5g yeast extract, add double distilled water to 1L, and sterilize at 121℃ for 20min.

[0022] Preparation of metabolically inactivated OP50: PFA (paraformaldehyde) was added to the OP50 bacterial solution with normal metabolism to a final concentration of 0.5%, and then returned to a 37°C shaking incubator for 1 hour; after 1 hour, the treated bacteria were transferred from the conical flask to a 50 mL centrifuge tube and centrifuged for 20 minutes, and the supernatant was discarded to obtain metabolically inactivated OP50 that inactivated bacterial replication and metabolism while maintaining cell structure and nutrients.

[0023] Example

[0024] PS-NPs single exposure: The exposure concentration of microplastics (20nm monodisperse polystyrene microspheres (PS-NPs)) was 10μg / L. PS-NPs solutions were prepared with normal metabolic OP50 bacterial solution and metabolically inactivated OP50 bacterial solution, respectively, and evenly spread on NGM plates. L1 stage Caenorhabditis elegans were randomly placed in the exposure plate, and 3 replicates were set up and cultured in a constant temperature incubator at 20℃.

[0025] Tamoxifen single exposure: The exposure concentration of tamoxifen was 10 μM. Tamoxifen citrate (Selleckchem, S1972) was dissolved in sterile dimethyl sulfoxide (DMSO) and added to NGM agar heated to 55°C at a final volume of 0.1% to obtain NGM plates containing tamoxifen at a final concentration of 10 μM. Normal metabolic OP50 bacterial solution and metabolically inactive OP50 bacterial solution were evenly spread on the NGM plates, respectively. L1 stage Caenorhabditis elegans were randomly placed in the exposure plates, and 3 replicates were set up and cultured in a constant temperature incubator at 20°C.

[0026] Combined exposure of PS-NPs and tamoxifen: The exposure concentration of microplastics was 10 μg / L. PS-NPs solution was prepared with normal metabolic OP50 bacterial solution and metabolically inactive OP50 bacterial solution, respectively, and evenly coated on NGM plates containing tamoxifen with a final concentration of 10 μM. L1 stage Caenorhabditis elegans were randomly placed in the exposure plate, and 3 replicates were set up and cultured in a constant temperature incubator at 20°C.

[0027] Head thrashes test of Caenorhabditis elegans: Collect the treated nematodes into an EP tube and rinse with K solution, then discard the supernatant. Place the clean nematodes in a small dish containing K solution, let it stand for 1 minute, and start counting the head thrashes for 1 minute. The center of the body is the X-axis, and the Y-axis is perpendicular to the X-axis. The nematode's head thrashes deviate from the X-axis and approach the Y-axis. Each completed round is recorded as a head thrashes. The calculation method is:

[0028]

[0029] Body bends test of C. elegans: Take a clean NGM plate, use a platinum wire pick to pick up the nematode and place it on the clean NGM plate. After the nematode adapts for 1 minute, start counting for 20 seconds. A complete S-shaped curve of the nematode is considered a body bend. The calculation method is:

[0030]

[0031] Pharyngeal pumping rate detection of C. elegans: Collect the exposed nematodes, wash them with K solution, move the clean nematodes to an empty NGM plate, and use a high-power microscope to observe the frequency of the pharyngeal pump beating within 1 minute. The calculation method is:

[0032]

[0033] Brood Size Test of C. elegans: Inoculate the C. elegans mother worms after exposure into NGM culture dishes and culture them. During the egg-laying period of the mother worms, transfer the mother worms to NGM culture dishes every day. After the old culture dishes containing eggs are cultured, count the number of C. elegans. Count the number of C. elegans in all culture dishes every day to obtain the number of offspring during the entire egg-laying period. The calculation method is:

[0034]

[0035] Body Size Detection of Caenorhabditis elegans: After the L1 stage Caenorhabditis elegans were exposed for 3 days, some nematodes were collected and washed, and the nematodes were placed in a water bath at 60°C for 10 minutes. The nematodes were taken out and transferred to solid culture medium, and the flat surface area of ​​the nematodes was measured using Image-ProH Express software. The calculation method is:

[0036]

[0037] All data in the present invention are expressed as mean ± standard deviation (SD). Graphs were drawn using GraphPad Prism10. Data analysis was performed using Microsoft Excel software for statistical analysis, and variance analysis was used to compare the differences between the two groups of data. Lifespan analysis was performed using a two-tailed two-sample T-test. All P values ​​less than 0.05 were statistically significant, *P<0.05, **P<0.01.

[0038] result:

[0039] like Figure 1 As shown, bacterial metabolic inactivation significantly alleviated the effect of PS-NPs (10 μg / L) exposure-enhanced tamoxifen (10 μM) toxicity on the reduction of body curvature in nematodes.

[0040] like Figure 2 As shown, bacterial metabolic inactivation significantly alleviated the effect of PS-NPs (10 μg / L) exposure-enhanced tamoxifen (10 μM) toxicity on the reduction of head bobbing in nematodes.

[0041] like Figure 3As shown, bacterial metabolic inactivation significantly alleviated the effect of PS-NPs (10 μg / L) exposure-enhanced tamoxifen (10 μM) toxicity on the reduction of pharyngeal pumping frequency in nematodes.

[0042] like Figure 4 As shown, bacterial metabolic inactivation significantly alleviated the effect of PS-NPs (10 μg / L) exposure on the enhanced tamoxifen (10 μM) toxicity on the reduction of nematode egg laying.

[0043] like Figure 5 As shown, bacterial metabolic inactivation significantly alleviated the effect of PS-NPs (10 μg / L) exposure-enhanced tamoxifen (10 μM) toxicity on the developmental delay of nematodes.

[0044] The above results revealed that when C. elegans was fed with normally metabolized OP50, exposure to PS-NPs alone at predicted environmentally relevant concentrations did not adversely affect the nematodes, while combined exposure to PS-NPs and tamoxifen adversely affected several toxic endpoints, including movement behavior, development, and reproduction. However, when C. elegans was fed with metabolically inactivated bacteria, protective effects on movement behavior, development, and reproduction were induced in the nematodes, indicating that metabolically inactivated bacteria can alleviate the toxic effects of PS-NP exposure that enhances tamoxifen, proposing a new perspective on the toxic effects of microorganisms on environmental pollutants and drugs.

Claims

1. Application of metabolically inactivated bacteria in the preparation of drugs for alleviating drug toxicity caused by nanoplastics.

2. The use according to claim 1, characterized in that: The drug toxicity caused by nanoplastics includes drug toxicity caused by the combination of nanoplastics and tamoxifen.

3. The use according to claim 2, characterized in that The metabolically inactivated bacteria is Escherichia coli OP50.

4. The use according to claim 3, characterized in that The method for obtaining the metabolically inactivated bacteria comprises: using paraformaldehyde to treat a live OP50 bacterial solution pre-shaken for 11 to 13 hours, and then applying the solution to an NGM culture medium for culturing after metabolic inactivation.

5. The use according to claim 1, characterized in that Under the treatment conditions of metabolically inactivated Escherichia coli OP50, the toxicity caused by nanoplastics in the concentration range of 0.1-10ug / L is reduced.

6. The use according to claim 1, characterized in that Under the conditions of metabolically inactivated bacteria treatment, the aggravated toxicity effect of 0.1-10μM tamoxifen caused by nanoplastic concentrations in the range of 0.1-10ug / L was alleviated.

7. A method for evaluating the effect of metabolically inactivated bacteria in alleviating drug toxicity caused by nanoplastics, characterized in that: (1) adding normal metabolic E. coli OP50 bacteria and metabolically inactivated E. coli OP50 bacteria to NGM solid culture medium to obtain NGM solid culture medium with different foods added; (2) NGM solid culture media containing different foods, including polystyrene nanoplastics, drugs, and a mixture of polystyrene nanoplastics and drugs, were prepared respectively, and L1 stage Caenorhabditis elegans were inoculated into these NGM solid culture media for cultivation. The effect of metabolically inactivated E. coli OP50 in alleviating the tamoxifen toxicity caused by nanoplastics was evaluated based on the head swing frequency, body movement frequency, pharyngeal pumping frequency, reproductive rate and development of the Caenorhabditis elegans.

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