Use of ppard gene in preparation of medicine for regulating mycotoxin toxicity

Abstract

The application discloses application of a PPARD gene in preparation of a medicine for regulating mycotoxin toxicity. The application firstly proposes that the PPARD gene or a coded protein thereof can be used as a target for relieving mycotoxin toxicity; the application firstly proposes that activation of the PPARD gene expression can reduce the toxic effect of mycotoxin, and overexpression of the PPARD gene at a cell level can relieve oxidative stress and tight connection damage induced by mycotoxin; and the application firstly proposes that Curcumin plays a role in relieving mycotoxin toxicity by activating the expression of the PPARD.

Description

Technical Field

[0001] This invention relates to the application of the PPARD gene in the preparation of drugs that regulate the toxicity of mycotoxins, and belongs to the field of mycotoxin biocontrol. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by molds and are widely found in mold-contaminated grains and feeds, posing a significant threat to human and animal health. With increasing public concern about feed safety and the resulting safety of livestock and poultry products, mycotoxin contamination has become a critical issue that urgently needs to be addressed in livestock and poultry farming. Among the known mycotoxin family, ochratoxin A (OTA) ranks second in toxicity and hazard, only after aflatoxin. OTA exhibits strong nephrotoxicity, hepatotoxicity, and enterotoxicity; OTA intake and accumulation can lead to organ damage. Existing research has primarily focused on OTA nephrotoxicity and hepatotoxicity, with limited research on its enterotoxicity. The intestine is the primary site of nutrient digestion and absorption, and is also the body's largest endocrine and immune organ. As a selective barrier, the intestinal barrier effectively prevents pathogenic microorganisms, toxins, and other exogenous harmful substances from entering the circulatory system; the integrity of the intestinal barrier plays a vital role in maintaining bodily health.

[0003] Although various mycotoxin detoxification methods exist, they suffer from numerous limitations, including high cost, low efficiency, and impact on feed nutritional value, making it difficult to completely eliminate the risk of livestock and poultry exposure to OTA. Finding a safe and effective method to control OTA toxicity is an urgent need in mycotoxin control, and biological methods are considered a promising strategy.

[0004] Peroxisome proliferator-activated receptor-δ / β (PPARD) is an important member of the nuclear receptor PPAR family, playing a regulatory role in fatty acid metabolism, obesity, wound healing, apoptosis, and inflammation. PPARD has already been used clinically as a target for the treatment of metabolic disorders such as dyslipidemia. Simultaneously, PPARD can protect epithelial cells from oxidative stress-induced apoptosis and inflammation by upregulating antioxidant enzymes. However, no studies have yet reported the regulatory effects and mechanisms of PPARD on mycotoxin toxicity. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide for the first time the application of the PPARD gene in the preparation of drugs that regulate the toxicity of mycotoxins, which can serve as a target for alleviating the toxicity of mycotoxins.

[0006] Technical solution: To solve the above technical problems, the present invention provides the application of the PPARD gene or its siRNA in the preparation of drugs that regulate the toxicity of mycotoxins, wherein the accession number of the PPARD gene is NM_001130241.3.

[0007] The present invention also provides the use of the PPARD gene or the protein encoded therein in the preparation of medicaments for the prevention and / or treatment of mycotoxin poisoning.

[0008] The present invention also provides the use of the PPARD gene or the protein encoded therein in the preparation of drugs to alleviate mycotoxin toxicity.

[0009] Among them, PPARD plays a role in mycotoxin detoxification by regulating oxidative stress and tight junctions between cells.

[0010] This includes overexpressing the PPARD gene or activating PPARD gene expression.

[0011] The method for activating PPARD gene expression includes using a PPARD activator.

[0012] The PPARD activator includes curcumin, the chemical formula of which is shown below:

[0013]

[0014] Furthermore, the activator exerts its detoxification effect on mycotoxins by increasing the expression of PPARD mRNA and protein.

[0015] The dosage forms of the drug include oral liquid, injection, tablet, pill, dispersant, capsule, drop pill, granule, suspension or emulsion.

[0016] The present invention also provides the use of curcumin in the preparation of medicaments for the prevention and / or treatment of mycotoxin poisoning.

[0017] The curcumin activates PPARD gene expression.

[0018] The dosage forms of the drug include oral liquid, injection, tablet, pill, dispersant, capsule, drop pill, granule, suspension or emulsion.

[0019] This invention also provides the application of peroxisome proliferator-activated receptor-δ / β as a target for mycotoxin detoxification. Furthermore, to overcome the shortcomings of existing technologies, this invention provides a natural compound that targets PPARD to prevent and treat mycotoxin poisoning, aiming to offer a new approach for the biological control of mycotoxin poisoning.

[0020] The present invention also provides a medicament capable of preventing and / or treating mycotoxin toxicity, said medicament being capable of activating the expression of the PPARD gene or protein.

[0021] The drug exerts its detoxifying effect on mycotoxins by increasing cell activity, inhibiting oxidative stress, and enhancing tight junctions between cells.

[0022] The drug also includes curcumin.

[0023] The present invention also provides a method for studying the function of genes related to mycotoxin detoxification, or screening drugs for preventing or treating mycotoxin enterotoxicity, or constructing mycotoxin exposure models, or analyzing drugs that target genes to alleviate mycotoxin toxicity, the method comprising overexpressing the PPARD gene, reducing PPARD gene expression, or activating PPARD gene expression.

[0024] Preferably, the overexpression of the PPARD gene includes constructing a PPARD overexpression vector using the pECMV-MCS-FLAG plasmid, the primer nucleotide sequences of which are shown in Table 1.

[0025] The method of reducing PPARD gene expression includes the use of siRNA, which includes si-PPARD-1, si-PPARD-2 and si-PPARD-3, and the nucleotide sequences of the siRNA are shown in Table 2.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention is the first to propose that the PPARD gene or its encoded protein can serve as a target for alleviating mycotoxin toxicity; 2. The present invention is the first to propose that activating PPARD gene expression can reduce the toxic effects of mycotoxins, and that overexpressing the PPARD gene at the cellular level can alleviate mycotoxin-induced oxidative stress and tight junction disruption; 3. The present invention is the first to propose that Curcumin can alleviate mycotoxin toxicity by activating PPARD expression. Attached Figure Description

[0027] Figure 1 The regulatory effect of PPARD overexpression on OTA cytotoxicity is shown in Figure A, where PPARD mRNA expression level changes in PPARD-overexpressing cells are shown in Figure B, the expression levels of tight junction proteins ZO-1, Occludin, and Cludin-1 in OTA-treated PPARD-overexpressing cells are shown in Figure C, and the oxidative stress level in OTA-treated PPARD-overexpressing cells is shown in Figure C. *** P<0.001;

[0028] Figure 2This study illustrates the activation effect of curcumin on PPARD expression. Figure A shows the PPARD mRNA expression level in cells treated with different concentrations of curcumin; Figure B shows the PPARD protein expression level in cells treated with different concentrations of curcumin. CUR: curcumin. ** P<0.01;

[0029] Figure 3 This study illustrates the regulatory effect of curcumin on OTA-induced cytotoxicity. Figure A shows the oxidative stress levels in different OTA and curcumin addition groups; Figure B shows the intracellular ROS levels in different treatment groups; Figure C shows the expression levels of tight junction proteins ZO-1, Occludin, and Cludin-1 in different OTA and curcumin addition groups; CUR: curcumin. ** P<0.01;

[0030] Figure 4 The effect of PPARD interference on curcumin's ability to alleviate OTA cytotoxicity is shown in Figure A, which illustrates the changes in PPARD mRNA expression levels in cells transfected with different siRNA fragments. Figure B shows the expression levels of tight junction proteins ZO-1, Occludin, and Cludin-1 in cells with PPARD interference after the addition of OTA and curcumin. CUR: curcumin. ** P<0.01, *** P<0.001. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] The main materials and reagents used in the examples are shown below:

[0033] (1) Experimental materials: IPEC-J2 cells (preserved in our laboratory); pECMV-MCS-FLAG (Wuhan Miaoling Biotechnology Co., Ltd., P0787).

[0034] (2) Experimental reagents: Ochratoxin A (OTA, Qingdao Prebang Biotechnology Co., Ltd.); Curcumin (MedChemExpress Biotechnology Co., Ltd., USA); DMEM high glucose medium (HyClone, USA); Fetal bovine serum, Opti-MEM medium (Thermo Fisher Scientific, USA); Penicillin-streptomycin mixture, reactive oxygen species detection kit, RIPA lysis buffer (Solepro Science & Technology Co., Ltd., Beijing); Cell Counting Kit-8 reagent kit (DOJINDO Technology Co., Ltd., Japan); Protease inhibitor and BCA protein content detection kit (Beyond Biotechnology Co., Ltd., Shanghai); 5×SDS-PAGE protein loading buffer and color pre-stained protein marker (Shanghai Yisheng Biotechnology Co., Ltd.); PVDF membrane (Millipore, USA); PAGE gel rapid preparation kit (Shanghai Yamei Biomedical Technology Co., Ltd.); PPARD (AF7800) (Beyond Biotechnology Co., Ltd., Shanghai); ZO-1 (ab221547) and Claudin-1 antibody (ab15098) (Abogen (Shanghai) Trading Co., Ltd.); Occludin (27260-1-AP) and GAPDH antibody (10494-1-AP) (Wuhan Sanying Biotechnology Co., Ltd.); Horseradish peroxidase-conjugated goat anti-mouse (HA1006) secondary antibody and goat anti-rabbit (HA1001) secondary antibody (Hangzhou Huaan Biotechnology Co., Ltd.); Ultrasensitive ECL chemiluminescence kit (Suzhou Xinsaimei Biotechnology Co., Ltd.); TRIzol RNA extraction reagent (Takara Bio Engineering (Dalian) Co., Ltd.); cDNA synthesis kit, fluorescence quantitative detection kit, high-fidelity DNA polymerase (P501), DNA purification and recovery kit, homologous recombination kit (Nanjing Novizan Biotechnology Co., Ltd.); DH5α competent cells (Beijing Qingke Biotechnology Co., Ltd.); endotoxin-free plasmid small-volume extraction kit (Beijing Tiangen Biotech Co., Ltd.); jetPRIME universal in vitro transfection reagent (Polyplus Biotechnology Co., Ltd., France).

[0035] The specific steps of the experimental method used in this embodiment are as follows:

[0036] 1. PPARD overexpression and interference vector construction

[0037] (1) Construction of PPARD overexpression vector

[0038] a. The gene sequence of PPARD (Gene ID: NM_001130241.3) was found in the NCBI database. Using pECMV-MCS-FLAG as the vector, primers were designed using homologous recombination. The CDS region of PPARD was amplified using high-fidelity DNA polymerase (the specific sequence after amplification is shown in SEQ ID NO: 1). The amplification system is shown in Table 1, and the reaction procedure is shown in Table 2. The primers used were synthesized by Nanjing Qingke Biotechnology Co., Ltd., and the specific primer sequences are shown in Table 3.

[0039] Table 1 PPARD amplification system

[0040]

[0041] Table 2 PPARD Amplification Reaction Procedure

[0042]

[0043] Table 3 Primer sequences for overexpression vectors

[0044]

[0045] b. After purifying and recovering the amplified product, ligate the PPARD gene amplification sequence to the linearized vector using homologous recombination according to the instructions. The ligation system (20 μL) consisted of: 0.03 pmol of pECMV-MCS-FLAG linearized vector, 0.06 pmol of insert fragment, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and ddH2O to a final volume of 20 μL. After gently mixing, the reaction solution was collected briefly at the bottom of the tube. The reaction was carried out in a 37°C metal bath for 30 min, and then immediately cooled on ice. The vector linearization enzyme digestion reaction system consisted of: 1 μg of pECMV-MCS-FLAG vector, 1 μL each of restriction endonucleases EcoRI and KpnⅠ, 5 μL of 10×NE Buffer (rCutSmart), and ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was digested at 37°C for 1 h.

[0046] c. Thaw competent cells on ice, add 10 μL of recombinant product to 100 μL of DH5α competent cells, gently pipette to mix, and incubate on ice for 30 min. After heat shock in a 42℃ metal bath for 45 s, immediately place on ice to cool for 2-3 min.

[0047] d. Add 900 μL of antibiotic-free LB medium and incubate at 37°C for 1 h (250 rpm). In a clean bench, use a sterile spreader to gently spread the mixture evenly onto ampicillin-resistant LB agar plates (containing 50 μg / mL ampicillin) and incubate upside down at 37°C for 14 h.

[0048] e. Pick a single colony, add ampicillin-resistant LB liquid medium (containing 50 μg / mL ampicillin), shake gently, and take 500 μL of bacterial solution for sequencing and identification of positive clones.

[0049] f. After expanding the positive clones using LB liquid medium containing ampicillin, recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit and NanoDrop. TM After passing quality inspection, the One micro spectrophotometer is stored at -80℃ for later use.

[0050] (2) Construction of interference carrier

[0051] Based on the porcine PPARD gene sequence (Gene ID: NM_001130241.3) in the NCBI database, BLOCK-iT was used. TM RNAi Designer software was used to design 3 pairs of siRNAs targeting the PPARD gene and 1 pair of negative controls (si-NC). The siRNAs were synthesized by Suzhou Genecasting Co., Ltd., and their sequence information is shown in Table 4.

[0052] Table 4. siRNA sequence information of the PPARD gene

[0053]

[0054] (3) Transfection

[0055] IPEC-J2 cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured until the density reached 70% before transfection. The transfection complex was prepared in a 1.5 mL sterile EP tube with the following composition: 100 μL jetPRIME Buffer, 2 μL siRNA / 2 μg plasmid, and 4 μL jetPRIME Reagent per well. The cells were vortexed for 10 s, briefly incubated at room temperature for 10 min, and then incubated with fresh medium after washing the cells with PBS. The transfection complex was then added to the corresponding wells, and the cells were cultured for 24 h to harvest RNA and 48 h to harvest protein. Overexpression and interference efficiencies were detected by qPCR and Western blot.

[0056] 2. Verification of overexpression efficiency

[0057] This invention uses qPCR to detect overexpression efficiency. The specific steps are as follows:

[0058] (1) Extraction of total RNA from cells

[0059] This study used the TRIzol method to extract total RNA from cells. The specific steps are as follows:

[0060] a. After washing the transfected IPEC-J2 cells with PBS, add 1 mL of TRIzol to each well, pipette thoroughly, and collect the cells in a 1.5 mL EP tube.

[0061] b. Add 200 μL of chloroform to each tube, vortex for 30 seconds, and centrifuge at 12800 rpm for 15 minutes at 4°C.

[0062] c. Pipette 400 μL of supernatant into a new 1.5 mL enzyme-free EP tube, add an equal volume of frozen isopropanol, mix by inverting the tube, and centrifuge at 12800 rpm for 10 min at 4 °C.

[0063] d. Discard the supernatant, retain the precipitate, add 1 mL of pre-cooled 75% ethanol, gently shake to wash away the sample precipitate, centrifuge at 12800 rpm for 10 min at 4℃; repeat twice, then remove as much residual ethanol as possible, open the lid and let stand for 3 min to allow the ethanol to evaporate completely.

[0064] e. Add 30 μL of RNase-free ddH2O and shake thoroughly to dissolve the RNA. After short-term infiltration, determine the RNA quality.

[0065] f. Use NanoDrop according to the instructions. TM After passing quality inspection, the One micro spectrophotometer is stored at -80℃ for later use.

[0066] (2) cDNA synthesis

[0067] Using total cellular RNA as a template, cDNA transformation was performed using a reverse transcription kit, with the following steps:

[0068] a. Genomic DNA removal: Prepare the premixed solution in an enzyme-free microcentrifuge tube according to the reaction system shown in Table 5, gently mix by pipetting, and react in a metal bath at 42°C for 2 min.

[0069] Table 5 gDNA Removal Reaction System

[0070]

[0071]

[0072] b. Reverse transcription reaction: Prepare the reaction mixture according to the system shown in Table 6, mix thoroughly by pipetting, and synthesize cDNA in a PCR instrument according to the program of 50℃ for 15 min and 85℃ for 5 s. After the reaction program is completed, store the cDNA sample at -20℃ for later use.

[0073] Table 6 Reverse transcription reaction system

[0074]

[0075] (3) qPCR detection

[0076] Using cDNA as a template, the reaction mixture was prepared according to the reaction system shown in Table 7. After vortexing and mixing, the mixture was added to a 96-well fluorescent quantitative plate, and the CT value was detected according to the reaction procedure shown in Table 8. GAPDH was used as an internal reference gene, and 2... -ΔΔCt The relative expression level of the target gene was analyzed by a method, and the primer information is shown in Table 9.

[0077] Table 7 qPCR reaction system

[0078]

[0079] Table 8 qPCR reaction procedure

[0080]

[0081] Table 9. qPCR primer information

[0082]

[0083] 3. Western blot detection of protein expression

[0084] (1) Total protein extraction and BCA concentration determination

[0085] After washing IPEC-J2 cells treated in the "PPARD overexpression and interference vector construction" step three times with PBS, RIPA lysis buffer was added to each well and the cells were lysed on ice for 30 min. After lysis, the lysis buffer and cell pellet were transferred to 1.5 mL centrifuge tubes using a cell scraper and centrifuged at 14000 rpm for 20 min. The supernatant was transferred to new centrifuge tubes for later use, and protein concentration was determined using the BCA Protein Assay Kit.

[0086] (2) Protein homogenization and protein denaturation

[0087] After standardizing the protein mass according to protein concentration and volume, 5×SDS-PAGE protein loading buffer was added, and the protein was denatured by heating in a 98℃ metal bath for 10 min. The denatured protein samples were then stored at -80℃ for later use.

[0088] (3) SDS-PAGE gel electrophoresis and membrane transfer

[0089] PAGE gels were prepared using a gel preparation kit. After the gels solidified, the corresponding samples were added, and electrophoresis was performed using Tris-Gly buffer. Electrophoresis was performed at a constant voltage of 80V for 30 minutes, followed by electrophoresis at 120V until the indicator band appeared at the bottom of the gel, at which point electrophoresis was stopped.

[0090] (4) Immunoblotting-transfer

[0091] Cut a PVDF membrane to the appropriate size according to the gel size, activate it with methanol, and then transfer it to the electrophoresis tank. Insert the transfer clamp into the transfer rack and place it in the electrophoresis tank. Add 500 mL of transfer buffer, cover with the safety cap, and turn on the power to start electrophoresis. The transfer conditions are constant current 300 A, 60 min.

[0092] (5) Blocking and antibody incubation

[0093] After transfer, the PVDF membrane was blocked with 5% skim milk powder at room temperature for 2 hours, washed three times with TBST, and then incubated with antibodies. The PVDF membrane was cut to size according to the protein bands and placed in an antibody incubation cassette. 10 mL of the corresponding primary antibody (PPARD, ZO-1, Claudin-1, Occludin, or GAPDH) diluted 1:1000 was added, and the membrane was incubated overnight on a shaker at 4°C. After primary antibody incubation, the antibody was recovered, washed three times with TBST, and then 10 mL of mouse and rabbit horseradish peroxidase-conjugated secondary antibody diluted 1:1000 was added. The membrane was incubated on a decolorizing shaker at room temperature for 2 hours, the antibody was recovered, and the membrane was washed three times with TBST before development.

[0094] (6) Development

[0095] After absorbing the TBST on the membrane with filter paper, it was placed in the developer solution and incubated in the dark for 2 minutes, and then the image was acquired using a Tianneng chemiluminescence imager.

[0096] 4. Cellular reactive oxygen species detection

[0097] IPEC-J2 cells were spaced at 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in 6-well plates and cultured for 12 h. After pretreatment with 2 μM curcumin (CUR) for 24 h, cells were cultured for another 48 h with 20 μM OTA. Cells were washed three times with PBS, digested with trypsin preheated to 37°C, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in 1 mL of DCFH-DA dilution buffer (DCFH-DA:Opti-MEM medium = 1:1000, v / v) and incubated at 37°C for 20 min, inverting to mix every 3 min. After incubation, cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was washed with PBS to remove unbound fluorescent probes. Finally, the cell pellet was resuspended in 300 μL of PBS. Flow cytometry was used to detect the excitation wavelength at 488 nm and the emission wavelength at 525 nm, and the results were analyzed using CytExpert software.

[0098] Example 1: High expression of PPARD can reduce OTA-induced cellular oxidative stress and intestinal barrier damage.

[0099] In this embodiment, the PPARD overexpression vector (PPARD-OE) was constructed according to the above method and transfected into IPEC-J2 cells. After 24 h, the cells were treated with 20 μM OTA for 48 h. The intracellular ROS level and the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 were detected. qPCR quantitative detection results showed that the PPARD mRNA level was significantly increased ( Figure 1 A) indicates that the overexpression vector was successfully expressed in cells. Furthermore, Western blot analysis revealed that PPARD overexpression significantly alleviated the inhibition of tight junction proteins ZO-1, Occludin, and Cludin-1 by OTA. Figure 1 B), and significantly reduced the OTA-induced increase in ROS levels ( Figure 1 C). The above results indicate that PPARD can alleviate OTA-induced intestinal barrier damage and oxidative stress, thereby reducing OTA-induced cytotoxicity.

[0100] Example 2: Curcumin targets and promotes the expression of PPARD

[0101] In this embodiment, a PPARD overexpression vector was constructed according to the above method, and transfected into IPEC-J2 cells. IPEC-J2 cells were treated with different doses of curcumin (0, 1, and 2 μM) for 24 h. qPCR and Western blot were used to detect changes in PPARD expression. It was found that curcumin could increase PPARD expression in mRNA (…). Figure 2 A) and protein levels ( Figure 2 B) Significantly promoted the expression of PPARD, indicating that PPARD can serve as a molecular target of curcumin.

[0102] Example 3: PPARD as a molecular target of curcumin reduces OTA-induced toxicity.

[0103] In this embodiment, a PPARD overexpression vector was constructed according to the above method, transfected into IPEC-J2 cells, and pretreated with curcumin for 24 h, followed by treatment with 20 μM OTA for 48 h. Flow cytometry was used to detect intracellular ROS levels in different treatment groups. Simultaneously, Western blot was used to detect changes in tight junction protein expression. The results showed that the addition of curcumin significantly reduced OTA-induced cellular oxidative stress levels (…). Figure 3 A and 3B). Western blot results showed that curcumin significantly alleviated the OTA-induced inhibition of expression of tight junction proteins ZO-1, Occludin, and Cludin-1. Figure 3 C).

[0104] Furthermore, PPARD expression in IPEC-J2 cells was interfered with using siRNA technology (IPEC-J2 cells were transfected with the siRNA sequences described in Table 2, and experiments were performed according to the method in Example 3). Western blot and qPCR validation showed a significant decrease in PPARD mRNA and protein expression levels, indicating that PPARD expression was successfully interfered with. Figure 4 A). In IPEC-J2 cells with disrupted PPARD expression, pretreatment with 2 μM curcumin for 24 h was followed by treatment with 20 μM OTA for 48 h. Western blot analysis revealed that disrupted PPARD expression exacerbated OTA-induced inhibition of tight junction proteins ZO-1, Occludin, and Claudin-1, and weakened the alleviating effect of curcumin on OTA-induced inhibition of these proteins. Figure 4 B).

[0105] The above results indicate that PPARD can serve as a molecular target of curcumin to alleviate OTA-induced oxidative stress and intestinal barrier disruption, thereby reducing the toxic effects of mycotoxins.

Claims

1. The application of a vector overexpressing the PPARD gene in the preparation of a drug to prevent ochratoxin A-induced porcine intestinal barrier damage, characterized in that, The nucleotide sequence of the CDS region of the PPARD gene is shown in SEQ ID NO.1.

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