Method for relieving vomitoxin-induced pig placental trophoblast cell injury by chlorogenic acid

Abstract

The invention discloses a method for relieving vomitoxin induced porcine placental trophoblast cell injury by chlorogenic acid, which optimizes the conditions of CGA treatment of porcine placental trophoblast pTr cells, including treatment concentration and treatment time, and evaluation of DON relieving effect. Important experimental materials and technical methods are provided for further relieving cytotoxicity caused by mycotoxin by using the CGA.

Description

Technical Field

[0001] The invention belongs to the technical field of biological antidotes, and particularly relates to a method for alleviating the damage of porcine placental trophoblast cells induced by vomitoxin induced by chlorogenic acid. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a common mycotoxin in feed that is extremely toxic to pigs. The reproductive toxicity of DON to pigs is mainly manifested in that DON can inhibit the proliferation of sow uterine cells and hinder the development of oocytes and embryos; cause oxidative damage to pig ovaries, morphological changes and apoptosis of ovarian granulosa cells, and inhibit cell proliferation; and cause structural damage to boar testicular supporting cells, cell cycle arrest, and oxidative stress. In addition, DON also has a toxic effect on pig placental trophoblast (pTr) cells, which is specifically manifested in decreased cell viability, inhibition of cell proliferation, decreased antioxidant capacity, and decreased migration ability.

[0003] Chlorogenic acid (CGA) is an acetal acid produced by caffeic acid and quinic acid (1-hydroxyhexahydrogallic acid). It is a phenyl acrylate polyphenol compound produced by plants through the shikimic acid pathway during aerobic respiration. Studies have shown that CGA can improve sperm motility and plasma membrane integrity in semen; improve the maturation, fertilization and developmental capacity of porcine oocytes during in vitro maturation, and reduce heat stress damage; improve porcine fertilized eggs and embryonic development. Therefore, CGA can improve the reproductive performance of pigs. There are currently no reports on the effects of CGA on porcine placental trophoblast cells.

[0004] The prior art (Dai Chaohui et al., Method for constructing a cell model of vomitoxin-induced porcine placental trophoblast cell injury, Application No.: 202411040253.1) mentions the toxic effect of DON on porcine placental trophoblast (pTr) cells, but does not provide a specific solution. The prior art (Yin Qingqiang et al., Application of chlorogenic acid compounds in the preparation of drugs for treating inflammation caused by DON, Application No.: 201911221469.7) mentions the protective effect of chlorogenic acid on inflammation and apoptosis of porcine intestinal epithelial cells induced by vomitoxin, but does not involve the effects on the proliferation, migration and antioxidant capacity of porcine placental trophoblast cells. In view of the important influence of early pregnancy embryo implantation on litter size, and the placental trophoblast cells directly determine the efficiency of embryo implantation, the present invention aims to evaluate the mitigation effect of CGA on DON-induced porcine placental trophoblast cell injury by exploring the safe concentration and treatment time of CGA, and provide new ideas and more experimental basis for using CGA to improve pig reproductive performance. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for using chlorogenic acid to alleviate the damage of porcine placental trophoblast cells induced by DON, optimize the conditions for treating porcine placental trophoblast cells with chlorogenic acid, including the safe concentration and treatment time of CGA, and evaluate the effect of CGA in alleviating the damage of porcine placental trophoblast cells induced by DON, which will provide new ideas and more experimental basis for using CGA to improve the reproductive performance of pigs.

[0006] In order to solve the above technical problems, an embodiment of the present invention provides a method for chlorogenic acid to alleviate the damage of porcine placental trophoblast cells induced by vomitoxin, comprising the following steps: Step 1: Inoculate porcine placental trophoblast cells into a 96-well plate, replace the culture medium, add CGA to treat the cells, and set the CGA concentration to: 0, 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL; the time is set to: 24h, 48h and 72h; after the treatment, add 10 μL of CCK-8 solution to each well, incubate at 37°C for 2h, detect the absorbance value at 450nm with an enzyme marker, calculate the cell survival rate, and screen out the safe dose and treatment time of CGA in pTr cells; Step 2: Inoculate the porcine placental trophoblast cells into a 96-well plate, replace the culture medium, and set up 4 treatment groups, including: NC control group, DON treatment group, CGA treatment group, and DON+CGA treatment group; wherein, the DON concentration was set to 0.5 μg / mL, and the CGA concentration was set to 50 μg / mL; the time was set to 48 h, and after the treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was detected by an ELISA instrument, and the cell survival rate was calculated to evaluate the effect of CGA on the alleviation of DON-induced inhibition of porcine placental trophoblast cell viability; Step 3: Inoculate porcine placental trophoblast cells into 12-well plates and set up four treatment groups, including NC control group, DON treatment group, CGA treatment group and DON+CGA treatment group; the DON concentration was set to 0.5 μg / mL, the CGA concentration was set to 50 μg / mL; the treatment time was set to 48 h; after the treatment, the total RNA of the cells was collected and reverse transcribed into cDNA. -ΔΔCt Methods The relative expression levels of genes were calculated; total cell proteins were collected, and protein expression levels were analyzed by Western blot to evaluate the effect of CGA on the proliferation inhibition, oxidative stress and migration inhibition of porcine placental trophoblast cells induced by DON; Step 4. Use the 12-well plate in step 3 to culture porcine placental trophoblast cells. When the cell density reaches 90%, use a 10 μL pipette tip to draw lines perpendicular to the bottom of the culture plate to make scratches. Count the scratch widths of each group of cells at 48 h to evaluate the effect of CGA in alleviating the inhibition of DON-induced porcine placental trophoblast cell migration ability, and obtain the method of CGA in alleviating DON-induced porcine placental trophoblast cell damage.

[0007] Among them, the porcine placental trophoblast pTr cells described in step 1 and step 2 are processed before the experiment: the porcine placental trophoblast pTr cells are inoculated into DMEM / F12 culture medium containing 10% fetal bovine serum, placed in a 37°C constant temperature incubator containing 5% CO2, and digested and passaged after full growth.

[0008] Wherein, in step 3, the gene expression level is expressed by detecting the expression level of the target gene in pTr cells of different treatment groups with CCND1, CDK4, SOD1, CAT and CEMIP as the target genes, and the expression of the target gene is normalized with GAPDH as the internal reference. In step 3, the protein expression level is expressed by detecting the expression level of the target protein in pTr cells of different treatment groups with CCND1, CDK4, SOD1, CAT and CEMIP as the target proteins, and the gray value of the target protein band is normalized with β-actin as the internal reference.

[0009] Among them, in step 4, the cell scratch healing rate is calculated using the following formula: Cell scratch healing rate (%) = [(scratch width value at T0 - scratch width value at Tt) / scratch width value at T0] × 100.

[0010] The beneficial effects of the above technical solution of the present invention are as follows: The present invention optimizes the method of chlorogenic acid to alleviate the damage of porcine placental trophoblast cells induced by vomitoxin, including the safe concentration of CGA and the time of treating cells, treating pTr cells with different concentrations of CGA, detecting the cell survival rate by CCK-8, and determining that the safe concentration of CGA for treating pTr cells is 25-75 μg / mL, and the treatment time is within 48 h. Using pTr cells in different treatment groups as target genes for CCND1, CDK4, SOD1, CAT and CEMIP, 2 -ΔΔCtMethods The relative expression level of the gene was calculated, the expression level of the protein was detected by western blot, and the migration ability of pTr cells was detected by cell scratch test to evaluate the mitigation effect of CGA on DON. The present invention determined that the mitigation effect of CGA was manifested in that CGA could alleviate the inhibition of pTr cell viability, proliferation inhibition, migration inhibition and decreased antioxidant capacity induced by DON, and finally determined that 50 μg / mL of CGA treated pTr cells for 48 hours could significantly alleviate DON-induced pTr cell damage, which would provide new ideas and more experimental basis for using CGA to improve pig reproductive performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a graph showing the pTr cell viability results after treatment with CGA at different times and concentrations in Example 1; Figure 2 This is a schematic diagram of how CGA alleviates the DON-induced inhibition of pTr cell viability in Example 2; Figure 3 Schematic diagram of the relative expression levels of proliferation genes, antioxidant genes and migration genes in cells of different treatment groups in Example 3; Figure 4 This is a schematic diagram of the relative expression levels of proteins in cells of different treatment groups in Example 3; Figure 5 This is a diagram showing the results of the cell scratch experiment in Example 4. DETAILED DESCRIPTION

[0012] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0013] Example 1: Safe concentration and time of CGA treatment of pTr cells 1. Main test reagents Porcine placental trophoblast (pTr) cells were preserved by our laboratory. CGA stock solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), culture medium DMEM / F12, trypsin-EDTA solution, and cell culture plates were purchased from Gibco (USA). Cell Counting Kit-8 was purchased from Shanghai Yamei Biopharmaceutical Technology Co., Ltd. (China).

[0014] 2. Cell culture pTr cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum in a 37°C incubator containing 5% CO2. After confluence, they were digested and passaged and inoculated into three 96-well plates. 15 treatment groups were set up: 3 time points (24h, 48h and 72h) and 5 CGA concentrations (0, 25μg / mL, 50μg / mL, 75μg / mL and 100μg / mL), and 3 replicates were set for each treatment.

[0015] 3. Cell Viability Assay After CGA treatment, 10 μL of CCK-8 solution was added to each well of the 96-well plate, and the absorbance at 450 nm was measured with a microplate reader after incubation for 2 h in an incubator. A standard curve was prepared according to the instructions, and then the viability of cells in each treatment well was calculated based on the standard curve.

[0016] 4. Statistical analysis The general linear model one-way analysis of variance (ANOVA) method of SPSS 16.0 software was used to compare the differences in cell viability among different treatment groups. Three replicates were set for each treatment, and the statistical results were expressed as "mean ± standard deviation" (x ± SD).

[0017] 5. Experimental results like Figure 1 As shown, when pTr cells were treated with different concentrations of CGA for 48 h, 50 μg / mL CGA significantly increased pTr cell viability (P<0.01), while 100 μg / mL CGA significantly decreased pTr cell viability (P<0.01). When pTr cells were treated with different concentrations of CGA for 72 h, 25 μg / mL CGA significantly increased pTr cell viability (P<0.01), 50 μg / mL CGA significantly increased pTr cell viability (P<0.01), while 100 μg / mL CGA significantly decreased pTr cell viability (P<0.01).

[0018] Example 2: CGA alleviates DON-induced inhibition of pTr cell viability 1. Main test reagents Porcine placental trophoblast (pTr) cells were preserved by our laboratory. DON and CGA were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), culture medium DMEM / F12, trypsin-EDTA solution, and cell culture plates were purchased from Gibco (USA). Cell Counting Kit-8 was purchased from Shanghai Yamei Biopharmaceutical Technology Co., Ltd. (China).

[0019] 2. Cell culture pTr cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum in a 37°C constant temperature incubator containing 5% CO2. After full growth, they were digested and subcultured and inoculated into a 96-well plate. Four treatment groups (NC control group, DON treatment group, CGA treatment group and DON+CGA treatment group) were set up. The DON concentration was set to 0.5μg / mL; the CGA concentration was set to 50μg / mL; the time was set to 48h; and 3 replicates were set for each treatment. Cell viability was detected after 48h.

[0020] 3. Cell Viability Assay After the treatment, add 10 μL of CCK-8 solution to each well of the 96-well plate cells, incubate in the incubator for 2 hours, and measure the absorbance at 450 nm with a microplate reader. Prepare a standard curve according to the instructions, and then calculate the viability of the cells in each treatment well based on the standard curve.

[0021] 4. Statistical analysis The differences in cell viability among different treatment groups were compared using one-way analysis of variance (ANOVA) with a general linear model using SPSS 16.0 software. Three replicates were set for each treatment, and the statistical results were expressed as “mean ± standard deviation” (x ± SD).

[0022] 5. Experimental results Figure 2 Schematic diagram of CGA alleviating DON-induced pTr cell viability inhibition in Example 2, wherein: Figure 2 A is the observation result of cells under microscope (100×); Figure 2 B is the statistical results of cell viability in different treatment groups. Figure 2 As shown in A, the cell states of pTr cells treated with different methods after 48 hours were significantly different: compared with the control group, the cells treated with DON showed obvious detachment and death, while the cells treated with CGA were in good condition, and even had a higher density than the NC group. It is worth noting that the pTr cell density in the DON+CGA group was significantly greater than that in the DON group. The CCK-8 results showed that ( Figure 2 B) Compared with the control group, the cell viability of the DON-treated group decreased significantly (P<0.01), and the cell viability of the CGA-treated group increased significantly (P<0.01); compared with the DON-treated group, the cell viability of the DON+CGA-treated group also increased significantly (P<0.01). Therefore, CGA alone and CGA and DON co-treatment of pTr cells can increase the viability of pTr cells, and CGA can alleviate the DON-induced inhibition of pTr cell viability.

[0023] Example 3 Effects of CGA and DON treatment on target gene expression in pTr cells 1. Main test reagents RNA extraction reagent Trizol, reverse transcription kit, and fluorescence quantification kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd. (Nanjing, China).

[0024] 2. Total RNA extraction and cDNA synthesis Strictly follow the instructions and use Trizol reagent to extract total RNA from cells in 12-well plates treated with DON. cDNA synthesis was performed using RNA as a template: 10 μL of the reaction system contained 2 μL of 5×qRT SuperMix II, 500 ng of total RNA, and RNasefree ddH2O to make up to 10 μL. Reaction conditions: 25℃ 10 min, 50℃ 30 min, 85℃ 5 min, and stored at 4℃.

[0025] qPCR primers were designed for the coding sequences of CCND1, CDK4, SOD1, CAT and CEMIP genes through an online website (https: / / primer3.ut.ee / ), and the expression of the target genes was normalized using GAPDH as an internal reference. The specific information of the primers used in this example is shown in Table 1.

[0026] Table 1 Amplification sequence primer information .

[0028] 3. Real-time fluorescence quantitative PCR Real-time fluorescence quantitative PCR reaction system: template cDNA 2.0 μL, upstream and downstream primers (10 μmol·L -1 ) 0.5µL each, 2×SYBR Premix ExTapTM II 10 µL, 50 × ROX Reference Dye II 0.5 µL, RNasefree ddH2O to a total volume of 20µL, 3 replicates for each sample. Amplification program: 95℃ 5 min; 95℃ 15s, 60℃ 34s, 40 cycles in total.

[0029] 4. Statistical analysis Use 2 -ΔΔCt Methods The relative expression levels of genes were calculated, and the internal reference gene GAPDH was used to normalize the gene expression levels. The general linear model one-way analysis of variance (ANOVA) method of SPSS 16.0 software was used to compare the differences in gene expression levels of cells in different treatment groups. Three replicates were set for each treatment, and the statistical results were expressed as "mean ± standard deviation" (x ± SD).

[0030] 5. Experimental results Figure 3 is the proliferation gene in the cells of different treatment groups in this Example 3 ( Figure 3 A) Antioxidant genes ( Figure 3 B) and migration genes ( Figure 3 C) Schematic diagram of relative expression levels. Figure 3 As shown in A, the qPCR results of the target genes CCND1 and CDK4 showed that compared with the control group, the expression levels of CCND1 and CDK4 genes in the DON treatment group were significantly or extremely significantly downregulated (P<0.05 or P<0.01); CGA treatment extremely significantly upregulated the expression levels of CCND1 and CDK4 genes (P<0.01); compared with the DON treatment group, the expression levels of CCND1 and CDK4 genes in the DON+CGA treatment group were extremely significantly upregulated (P<0.01).

[0031] like Figure 3 As shown in B, the qPCR results of the target genes were antioxidant genes CAT and SOD1. Compared with the control group, the expression levels of CAT and SOD1 genes in the DON treatment group were significantly or extremely significantly downregulated (P<0.05 or P<0.01); CGA treatment had no significant effect on the expression levels of CAT and SOD1 genes (P>0.05); compared with the DON treatment group, the expression levels of CAT and SOD1 genes in the DON+CGA treatment group were significantly or extremely significantly upregulated (P<0.05 or P<0.01).

[0032] like Figure 3 As shown in C, the qPCR results of the target gene CEMIP showed that compared with the control group, the expression level of the CEMIP gene in the DON treatment group was extremely significantly downregulated (P<0.01); CGA treatment extremely significantly upregulated the expression level of the CEMIP gene (P<0.01); compared with the DON treatment group, the expression level of the CEMIP gene in the DON+CGA treatment group was extremely significantly upregulated (P<0.01).

[0033] Example 4: Effects of CGA and DON treatment on the expression of target protein in pTr cells 1. Main test reagents Protein extraction kit Protein Extraction Kit, protein concentration determination kit BCA ProteinAssay Kit, SDS-PAGE gel preparation kit and ECL chemiluminescence developer were purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd. (Shanghai, China). Protein antibodies primary antibodies (CCND1, CDK4, SOD1, CAT, CEMIP and β-actin) and secondary antibodies (HRP-IgG) were purchased from Abimat Pharmaceutical Technology (Shanghai) Co., Ltd. (China).

[0034] 2. Total cell protein extraction and concentration determination Cell treatment was the same as in Example 3. After cell treatment, total cell protein was extracted using a Protein Extraction Kit in strict accordance with the instructions, and protein concentration was detected using a BCA Protein Assay Kit, a standard curve was drawn using 5 standards and corresponding OD values, and then the protein concentration was calculated by detecting the OD value of the protein sample at 450 nm.

[0035] 3. Western Blot Take 20μg protein sample, add buffer, boil in boiling water for 10min to denature the protein, and then perform SDS-PAGE gel electrophoresis: 10% gel, 80V voltage for the upper gel, 100V voltage for the lower gel, and electrophoresis for 90-120min. After the steps of transfer, blocking, incubation with primary antibodies (CCND1, CDK4, SOD1, CAT, CEMIP and β-actin), washing, incubation with secondary antibodies, washing, etc., ECL chemiluminescence development was used, and finally the expression of the target protein was analyzed using a gel image processing system.

[0036] 4. Statistical analysis The differences in gene expression levels in cells of different treatment groups were analyzed using general linear model multivariate analysis using SPSS 16.0 software. Three replicates were set for each treatment, and the statistical results were expressed as "mean ± standard deviation" (x ± SD).

[0037] 5. Experimental results Figure 4 Schematic diagram of the relative expression levels of proteins in cells of different treatment groups in Example 3, wherein: Figure 4 A is a protein immunoblot band; Figure 4 B is the statistical result of the gray value of the protein band. Figure 4 As shown, DON significantly reduced the expression levels of proteins related to proliferation, migration and anti-oxidation (P<0.05 or P<0.01). Compared with the DON group, the protein expression levels in pTr cells of the DON+CGA group were significantly or extremely significantly increased (P<0.05 or P<0.01).

[0038] Embodiment 5 1. Cell scratch assay to detect the migration ability of pTr cells Four treatment groups of pTr cells (NC control group, DON treatment group, CGA treatment group and DON+CGA treatment group) were cultured in 12-well plates. After the cell density reached 90%, a 10μL pipette tip was used to draw a line perpendicular to the bottom of the culture plate to make a scratch. The scratch width of cells in different treatment groups was counted at 48h. The cell scratch healing rate was calculated according to the formula: Cell scratch healing rate (%) = [(scratch width value at T0-scratch width value at Tt) / scratch width value at T0] × 100.

[0039] 2. Statistical analysis The differences in scratch healing rates of cells in different treatment groups were compared using the t-test provided by SPSS 16.0 software. Three replicates were set for each treatment, and the statistical results were expressed as "mean ± standard deviation" (x ± SD).

[0040] 3. Experimental results Figure 5 This is a graph showing the results of the cell scratch test in Example 4, wherein: Figure 5 A is the result of cell scratch microscopy observation (100×); Figure 5 B is the statistical result of cell scratch healing rate. Figure 5 As shown in the figure, there were significant differences in the cell states of pTr cells treated with different methods after 48 hours: compared with the control group, cells treated with DON showed obvious detachment and death, while cells treated with CGA were in good condition, and even had a higher density than the NC group. In addition, the pTr cell density in the DON+CGA group was significantly greater than that in the DON group. The statistical results also showed that the cell healing percentage in the DON-treated group was significantly lower than that in the control group (P<0.01).

[0041] In summary, the present invention proposes a method for CGA to alleviate DON-induced porcine placental trophoblast cell damage, and CGA can alleviate DON-induced pTr cell viability decline, proliferation inhibition, migration inhibition and antioxidant capacity decline. In the present invention, the optimal condition for CGA treatment of pTr cells is 50 μg / mL CGA treatment of pTr cells for 48 h.

[0042] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for alleviating the damage of porcine placental trophoblast cells induced by vomitoxin by chlorogenic acid, characterized in that: The following steps are involved: Step 1: Inoculate porcine placental trophoblast cells into a 96-well plate, replace the culture medium, add CGA to treat the cells, and set the CGA concentration to: 0, 25 μg / mL, 50 μg / mL, 75 μg / mL and 100 μg / mL; the time is set to: 24h, 48h and 72h; after the treatment, add 10 μL of CCK-8 solution to each well, incubate at 37°C for 2h, detect the absorbance value at 450nm with an enzyme marker, calculate the cell survival rate, and screen out the safe dose and treatment time of CGA in pTr cells; Step 2: Inoculate the porcine placental trophoblast cells into a 96-well plate, replace the culture medium, and set up 4 treatment groups, including: NC control group, DON treatment group, CGA treatment group, and DON+CGA treatment group; wherein, the DON concentration was set to 0.5 μg / mL, and the CGA concentration was set to 50 μg / mL; the time was set to 48 h, and after the treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was detected by an ELISA instrument, and the cell survival rate was calculated to evaluate the effect of CGA on the inhibition of DON-induced porcine placental trophoblast cell viability. Step 3: Inoculate porcine placental trophoblast cells into 12-well plates and set up four treatment groups, including NC control group, DON treatment group, CGA treatment group and DON+CGA treatment group; the DON concentration was set to 0.5 μg / mL, the CGA concentration was set to 50 μg / mL; the treatment time was set to 48 h; after the treatment, the total cell RNA was collected and reverse transcribed into cDNA. -ΔΔCt Methods The relative expression levels of genes were calculated; total cell proteins were collected, and protein expression levels were analyzed by Western blot to evaluate the effect of CGA on the proliferation inhibition, oxidative stress and migration inhibition of porcine placental trophoblast cells induced by DON; Step 4. Use the 12-well plate in step 3 to culture porcine placental trophoblast cells. When the cell density reaches 90%, use a 10 μL pipette tip to draw lines perpendicular to the bottom of the culture plate to make scratches. Count the scratch widths of each group of cells at 48 h to evaluate the effect of CGA in alleviating the inhibition of DON-induced porcine placental trophoblast cell migration ability, and obtain the method of CGA in alleviating DON-induced porcine placental trophoblast cell damage.

2. The method of chlorogenic acid for alleviating porcine placental trophoblast cell damage induced by vomitoxin according to claim 1, characterized in that: The porcine placental trophoblast cells described in step 1 and step 2 were processed before the experiment: the porcine placental trophoblast cells were inoculated into DMEM / F12 culture medium containing 10% fetal bovine serum, cultured in a 37°C constant temperature incubator containing 5% CO2, and digested and passaged after full growth.

3. The method of chlorogenic acid for alleviating porcine placental trophoblast cell damage induced by vomitoxin according to claim 1, characterized in that: In step three, the gene expression level is expressed by detecting the expression level of the target gene in pig placental trophoblast cells of different treatment groups with CCND1, CDK4, SOD1, CAT and CEMIP as target genes, and the expression of the target gene is normalized with GAPDH as an internal reference; in step three, the protein expression level is expressed by detecting the expression level of the target protein in pig placental trophoblast cells of different treatment groups with CCND1, CDK4, SOD1, CAT and CEMIP as target proteins, and the grayscale value of the target protein band is normalized with β-actin as an internal reference.

4. The method of chlorogenic acid for alleviating porcine placental trophoblast cell damage induced by vomitoxin according to claim 1, characterized in that: In step 4, the cell scratch healing rate is calculated using the following formula: Cell scratch healing rate (%) = [(scratch width value at T0 - scratch width value at Tt) / scratch width value at T0] × 100.

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