CN115120727A - Application of S100A9 inhibitor in preparation of drugs for preventing and treating C-type clostridium perfringens infectious diarrhea - Google Patents
Application of S100A9 inhibitor in preparation of drugs for preventing and treating C-type clostridium perfringens infectious diarrhea Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
The invention discloses application of an S100A9 inhibitor in preparation of a medicine for preventing and treating C-type clostridium perfringens infectious diarrhea. The research of the invention discovers that the expression quantity of S100A9 in diarrhea tissues and cells of C-type clostridium perfringens infected piglets is increased, S100A9 aggravates inflammation injury of pig intestinal epithelial cells induced by C-type clostridium perfringens infection, inhibits cell viability and proliferation, destroys intercellular tight connection, and can be used as a medicament for treating C-type clostridium perfringens infected diarrhea by down-regulating the S100A9 gene. The invention also provides a medicine for treating the clostridium perfringens type C infectious porcine diarrhea. After C-type clostridium perfringens infection, the medicine can improve the activity of porcine intestinal epithelial cells, improve the proportion of S-phase cells and promote cell proliferation; attenuating the cytotoxicity of clostridium perfringens type C infection; improves the tight connection of cells and has good prevention and treatment effect on the C-type clostridium perfringens infectious diarrhea.
Description
Technical Field
The invention belongs to the field of livestock disease medicines, and particularly relates to an application of an S100A9 inhibitor in preparation of a medicine for preventing and treating C-type clostridium perfringens infectious diarrhea.
Background
The S100 protein family is a large calcium binding protein family, the S100 protein is a protein with high affinity to calcium ions and is firstly separated from bovine brain tissue, in recent years, with the further research, more types of S100 proteins are discovered, more than 20 members of the S100 family exist at present, and the S100A9 protein is one of the important proteins. The S100A9 protein is obtained by purifying myeloid cells at first, and the S100A9 protein is related to various diseases, such as the expression of the S100A9 gene in various tumors, including liver cancer, lung cancer, prostatic cancer and the like; in immune diseases, the S100A9 protein induces cell proliferation and asthma attack; drugs for treating diseases by utilizing the S100A9 characteristics are also developed successively; pimecrolimus prevents atopic dermatitis by inducing up-regulation of S100A8/A9 and other gene expression; in the treatment of cardiovascular disease, some scholars attempt to treat atherosclerosis by down-regulating the inflammatory pathway by blocking S100A8/a9 gene expression with drugs.
The clostridium perfringens type C is a pathogenic bacterium causing clostridium enteritis of pigs, can cause necrotic enteritis of piglets, and has the characteristics of short course of disease and high mortality. The existing medicine for treating clostridium perfringens type C causes the clostridial enteritis of pigs mainly comprises the following components: antibiotics, vaccines, and the like. Although the incidence rate of diarrhea of piglets is reduced by means of antibiotics, vaccines and the like, the healthy growth and meat quality of the piglets are greatly damaged by excessive use of the antibiotics; the Chinese invention patent discloses a C-type clostridium perfringens toxoid vaccine for piglet red dysentery, and relates to a C-type clostridium perfringens toxoid vaccine for preventing and treating diseases generated by the vaccine; however, immunotherapy has individual differences, and different individuals have different immune responses to vaccines, and often need multiple vaccinations, and whether antibodies are produced is determined, and only the individuals producing antibodies will produce an immune response to C-type clostridium perfringens toxin, which is seen to have certain deficiencies and risks in effectiveness. Therefore, there is a need to develop a new drug against clostridium perfringens type C infectious diarrhea.
Disclosure of Invention
The invention provides an application of an S100A9 inhibitor in preparing a medicine for preventing and treating C-type clostridium perfringens infectious diarrhea, aiming at solving the problems in the prior art.
The invention aims to provide application of an S100A9 inhibitor in preparation of a medicine for preventing and treating C-type clostridium perfringens infectious diarrhea.
The invention also aims to provide a medicament for preventing and treating diarrhea, which comprises the S100A9 inhibitor.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of an S100A9 inhibitor in preparation of a medicine for preventing and treating C-type clostridium perfringens infectious diarrhea.
Experiments show that the expression quantity of S100A9 in diarrhea tissues and cells of C-type clostridium perfringens infected piglets is increased, S100A9 aggravates inflammation injury of pig intestinal epithelial cells induced by C-type clostridium perfringens infection, inhibits cell activity and cell proliferation, destroys intercellular tight connection, and through inhibiting S100A9, the down regulation of S100A9 gene can be used as a medicine for treating C-type clostridium perfringens infected diarrhea.
Further, the S100a9 inhibitor is an inhibitor of the expression of the S100a9 gene in tissues and/or intestinal epithelial cells.
Further, the tissue is visceral tissue.
Further, the visceral tissue is lung, spleen, liver, ileum, kidney, duodenum, jejunum or heart.
Further, the S100A9 inhibitor reduces the expression levels of inflammatory factors IL-6, IL-8, TNF-alpha and IL-1 beta in intestinal epithelial cells infected by C-type clostridium perfringens, and reduces cell damage.
Further, the S100a9 inhibitor maintains cell viability of the intestinal epithelial cells under clostridium perfringens type C infection.
Further, the S100A9 inhibitor increases the S phase ratio of the intestinal epithelial cells infected by the clostridium perfringens type C and maintains the cell proliferation of the intestinal epithelial cells.
Further, the S100a9 inhibitor reduces the activity of reactive oxygen species and LDH enzymes of clostridium perfringens type C infected lower intestinal epithelial cells, thereby reducing the cytotoxicity of clostridium perfringens type C against intestinal epithelial cells.
Further, the S100a9 inhibitor maintains significant upregulation of expression of zon-1, OCLN, and CLDN-12, thereby inhibiting decreased enterocyte tight junction induced by clostridium perfringens type C infection.
The invention also claims a medicament containing the S100A9 inhibitor for preventing and treating diarrhea.
Further, the diarrhea is clostridium perfringens type C infectious diarrhea.
Further, the S100a9 inhibitor is an RNA interference drug that inhibits transcription of the S100a9 gene.
Further, the S100A9 inhibitor is interfering RNA si-S100A9, and the sequence of the interfering RNA is shown as SEQ ID No. 21-SEQ ID No. 22.
Further, the S100A9 inhibitor is a monoclonal antibody or a polyclonal antibody for inhibiting S100A9 protein translated from S100A9 gene.
Furthermore, the medicine also contains necessary auxiliary materials.
The scheme of the invention is suitable for C-type clostridium perfringens infectious diarrhea, and is suitable for mammals such as pigs, cows, horses, sheep and humans, as well as poultry and the like, and animals which are easy to be infected by the C-type clostridium perfringens, especially young animals.
The invention has the beneficial effects that:
(1) the invention provides application of an S100A9 inhibitor in preparation of a medicine for treating C-type clostridium perfringens infectious diarrhea, and also provides a novel medicine for preventing and treating diarrhea.
(2) After clostridium perfringens type C infection, drugs containing the S100a9 inhibitor can improve intestinal epithelial cell viability, increase S-phase cell proportion, promote cell proliferation; reducing the toxicity of clostridium perfringens type C infection on cells; improves the tight connection among cells and can play a good role in preventing and treating C-type clostridium perfringens infectious diarrhea.
Drawings
FIG. 1 is a graph of the relative mRNA expression levels of S100A9 in different tissues of piglets.
FIG. 2 is a graph showing the relative expression levels of S100A9 protein in ileal tissues; IC in the figure represents a control group; IS represents a susceptible group; IR means tolerance group.
FIG. 3 is the expression level of S100A9 protein in jejunal tissue; JC in the figure represents the control group; JS represents the susceptible group; JR denotes the tolerability group.
FIG. 4 is the expression of S100A9 over time under CPB2 toxin treatment conditions.
FIG. 5 shows the transfection efficiency of S100A 9; in the figure, A represents the relative mRNA expression level of S100A 9; b represents the protein relative expression level of S100a 9.
FIG. 6 is a graph of the effect of S100A9 on CPB 2-induced expression of IPEC-J2 cytokine; in the figure, A represents the relative mRNA expression level; b represents the relative expression level of the protein.
FIG. 7 is a graph of the effect of S100A9 on CPB2 toxin-induced IPEC-J2 cell viability and proliferation; in the figure, A represents the cell viability measured by the CCK8 method; b represents EDU-positive cell ratio; c represents EDU cell proliferation assay.
FIG. 8 is a CPB 2-induced measurement of reactive oxygen species levels and cytotoxicity in IPEC-J2 cells; panel a represents ROS level detection; b represents detection of Lactate Dehydrogenase (LDH) activity.
FIG. 9 is a graph of the effect of S100A9 on the CPB2 toxin-induced IPEC-J2 cell cycle.
FIG. 10 is a graph of the effect of S100A9 on CPB2 toxin-induced expression of IPEC-J2 cell claudin.
The above figures are labeled: "x" indicates significance P <0.01, "x" significance P <0.05, "ns" indicates significance P > 0.05.
Detailed Description
The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Experimental example 1 animal experiments S100A9 expression in tissues
1. Experimental methods
(1) Experiment grouping
Randomly selecting 30 healthy piglets (long white pigs, male pigs and large white pigs, female pigs and male pigs) with the age of 7 days, wherein the weight of each piglet is 3.2Kg +/-0.5 Kg, grouping the piglets, randomly selecting 5 piglets as a control group, 25 piglets as an experimental group, and drenching 5mL of C-type clostridium perfringens to each piglet of the experimental group, wherein the colony forming unit of drenching the C-type clostridium perfringens is 1 multiplied by 10 9 CFU/mL, continuously drenching for 5 days, and the drenching amount is the same every day. The experimental groups were divided into sensitive and tolerant groups according to stool scores. After treatment, the tissues of heart, liver, spleen, lung, kidney, duodenum, jejunum and ileum of each treatment group are taken and rapidly frozen and stored by liquid nitrogen, and the animal experimental method conforms to the ethical standard and international convention of human beings.
(2) RT-qPCR detection of expression level of S100A9
The expression level of S100A9 in Lung (Lung), Spleen (Spleen), Liver (Liver), Ileum (Ileum), Kidney (Kidney), Duodenum (Duodenum), Jejunum (Jejunum) and Heart (Heart) tissues of control, susceptible and tolerant piglets was determined by Real-Time Quantitative fluorescence (RT-qPCR), total RNA of piglet tissues was extracted from the tissues by TRIzol (TIANGEN, Beijing, China) reagent according to the instructions of TRIzol kit, GAPDH was used as internal reference, and the corresponding primers were shown in Table 1. Qualified RNA was detected, and cDNA was synthesized using the Evo M-MLV reverse transcription master mix kit (Accurate Biotechnology, Hunan, China). RT-qPCR in LightCycler480II Instrument (Roche Switzerland) and
green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China), RT-qPCR assay S100A9 primers are shown in Table 1, and the primer sequences are SEQ ID No. 1-SEQ ID No.2 and SEQ ID No. 11-SEQ ID No. 12.
TABLE 1 primers required for RT-qPCR detection
(3) Western blot detection
Protein expression levels of S100A9 in ileum and jejunum tissues were examined by Western Blot. Specifically, a proper amount of lysis buffer (RIPA) containing phenylmethanesulfonyl fluoride (PMSF) is adopted to collect the total protein of ileum and jejunum tissues, a BCA protein detection kit is adopted to determine the protein concentration, the denatured protein sample is subjected to electrophoresis on 8% SDS-PAGE, the gel voltage is concentrated to be 75V, and the gel separation is 120V. The membrane was sealed with 5% skim milk (0.5% TBST buffer) for 1h at 37 ℃ with shaking, then diluted with S100a9 antibody at 1:900 (recommended dilution ratio 1: 800-1: 1000) or β -actin antibody at 1:900 (recommended dilution ratio 1: 800-1: 1000) overnight at 4 ℃, then combined with horseradish peroxidase (HRP) -labeled goat anti-mouse secondary antibody at 1:900 (recommended dilution ratio 1: 800-1: 1000), washed 3 times with TBST on a decolourizing shaker, exposed using a chemiluminescence detection system, and quantitatively analyzed using ImageJ (v1.8.0) software.
2. Results of the experiment
The expression level of S100A9 in the diarrhea piglets is increased, the experimental result is shown in figure 1, and the expression level of S100A9 in the susceptible group is obviously higher than that in the control group. Based on this feature, we further identified the small intestine tissue most closely related to the occurrence of diarrhea in piglets, and examined the protein expression level of S100a9 in ileum (fig. 2) and jejunum (fig. 3) tissues by Western Blot method. The result shows that the expression trend of the gene is consistent with that of RT-qPCR.
Experimental example 2 expression of S100A9 in cells
1. Experimental method
(1) Cell culture
The porcine IPEC-J2 cell line (porcine small intestine epithelial cell line) was provided by beijing beina biotechnology limited. All cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, USA) Medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin and streptomycin. Cells were incubated at 37 ℃ with 5% CO 2 The environment incubator is used for culture. When the confluency of the cells reached more than 80%, the cells were digested with a 0.25% trypsin solution and subcultured or tested.
(2) Cell infection with CPB2 toxin
The IPEC-J2 cells obtained by the culture in the step (1) are averagely divided into 2 groups, a CPB2 toxin treatment group and a control group, the CPB2 toxin treatment group with the dosage of 20ug/mL is added into the CPB2 toxin treatment group, the control group is given with the corresponding dosage of normal saline for treatment, the expression of S100A9 is measured at 0h, 12h, 24h, 36h and 48h respectively, and the measuring method is the same as the RT-qPCR method of S100A9 expression in piglet tissues in the test example 1.
2. Results of the experiment
As shown in fig. 4, it can be seen that the CPB2 toxin-treated group significantly induced IPEC-J2 cell damage from 12h, and the expression of S100a9 was time-dependently up-regulated and peaked at 24h after CPB2 treatment and then stabilized. The level of S100a9 was significantly increased in CPB2 group compared to the control group; the results indicate that S100a9 expression is also significantly elevated in CPB2 toxin-induced IPEC-J2 cells, indicating that S100a9 plays a key role in CPB2 toxin-induced IPEC-J2 cell injury.
Experimental example 3 transfection efficiency of S100A9 into IPEC-J2 cells
1. Experimental methods
Inoculating the cell suspension onto a culture plate, and starting transfection when the cell growth state is good and the cell confluency reaches 70-80%. Dividing cells to be transfected into 4 groups, namely an interference negative control group (si-NC), an S100A9 inhibitor (si-S100A9) group, an mRNA overexpression empty vector group (pcDNA3.1) and an S100A9 overexpression vector group (pc-S100A9), wherein each group is respectively subjected to overexpression by using an interference negative control (si-NC) vector, an S100A9 inhibitor vector and mRNATransfection of IPEC-J2 cells with empty vector and S100A9 overexpression vector, and reference to knockout and overexpression of S100A9
2000Reagent
(Invitrogen, CA, USA) transfection reagent Specification, vector construction based on pcDNA3.1 cloning vector, 5'NheI and 3' XhoI as cloning sites, S100A9 overexpression vector was constructed, named pc-S00A9, and synthesized by Kingzhi Biotech, Suzhou. Interference negative controls (si-NC) and S100A9 inhibitors (si-S100A9) were both synthesized by Shanghai Jima pharmaceutical technology, Inc. (Shanghai, China). Wherein, the sequence information of the interfering RNA is shown in Table 2, and the primer sequences are SEQ ID No. 19-SEQ ID No. 22. And (3) starting transfection when the inoculated cells are cultured until the confluence reaches 70% -80%, adding CPB2 toxin into each group after 24h of transfection to ensure that the toxin concentration is 20 mu g/ml, and continuing culturing for 24 h. After completion of the culture, the expression of S100A9 was examined by RT-qPCR and Western Blot method in the same manner as in the RT-qPCR and Western Blot method for S100A9 expression in the tissues of piglets in test example 1.
TABLE 2 sequence information of interfering RNAs
2. Results of the experiment
The experimental results are shown in FIG. 5, and the RT-qPCR results show that the expression level of S100A9 is obviously increased after the transfection of pc-S100A9 compared with the overexpression negative control pcDNA3.1. Expression levels of S100a9 were significantly reduced after si-S100a9 transfection compared to the interference negative control si-NC. The detection result of Western Blot is consistent with the detection result of RT-qPCR, which indicates that the overexpression and interference of S100A9 are successful, and subsequent tests can be carried out.
Experimental example 4 relationship between S100A9 and CPB2 toxin-induced inflammatory factor
1. Experimental methods
Inoculating the cell suspension onto a culture plate, and starting transfection when the cell growth state is good and the cell confluence reaches 70% -80%. Dividing cells to be transfected into 6 groups, namely a Control group (Control), a CPB2 treatment group (CPB2), a negative Control group (CPB2+ pcDNA3.1), an S100A9 overexpression group (CPB2+ pc-S100A9), an interference negative Control group (CPB2+ si-NC) and an S100A9 interference group (CPB2+ si-S100A 9); the Control group (Control) is not treated, CPB2 toxin is added into the CPB2 treatment group (CPB2) to enable the concentration of the toxin to be 20 mug/ml, other 4 groups are respectively transfected by an mRNA over-expression empty vector, an S100A9 over-expression vector, an S100A9 inhibitor negative Control (si-NC) vector and an S100A9 inhibitor vector into IPEC-J2 cells, then CPB2 toxin with the concentration of 20 mug/ml is added, the cells are continuously cultured for 24h, after the culture is finished, the mRNA relative expression conditions of IL6, IL8, TNF alpha and IL-1 beta are detected by adopting an RT-qPCR method, and the determination method refers to the RT-qPCR method in test example 1, wherein the primer sequences corresponding to IL6, IL8, TNF alpha and IL-1 beta and the total mRNA are shown in Table 1, and the primer sequences are SEQ ID No. 3-SEQ ID No. 12.
And simultaneously, enzyme-linked immunosorbent assay (ELISA) is adopted to detect the levels of inflammatory factors IL6, IL8, TNF alpha and IL-1 beta in the cells of each group so as to verify the detection accuracy of RT-qPCR. The specific operation is as follows: cell culture supernatant after transfection and CPB2 toxin treatment for 48h was collected and centrifuged at 2500rpm for 20min to obtain a test sample. According to the ELISA kit specification, the contents of inflammatory cytokines IL6, IL8, TNF alpha and IL-1 beta in a sample to be tested were detected by using an ELISA kit (Jiangsu Kott Biotechnology Co., Ltd., Jiangsu, China). Finally, OD450 values are measured under a microplate reader, a standard curve is drawn, and the concentration of the sample is calculated and expressed in pg/mL.
2. Results of the experiment
RT-qPCR results are shown in FIG. 6A, and after the CPB2 toxin induces IPEC-J2 cells, the expressions of inflammatory factors IL6, IL8, TNF alpha and IL-1 beta are all significantly up-regulated. After overexpression of S100A9, CPB2 induced expression of IL6, IL8, TNF alpha and IL-1 beta in IPEC-J2 cells is all significantly up-regulated; CPB 2-induced expression of IL6, IL8, TNF alpha and IL-1 beta in IPEC-J2 cells under interference of S100A9 was all significantly reduced. Meanwhile, enzyme-linked immunosorbent assay (ELISA) is adopted to detect the content of inflammatory factors IL6, IL8, TNF alpha and IL-1 beta in cell culture supernatant, as shown in figure 6B, the determination result is consistent with the RT-qPCR result. The above results indicate that S100a9 can increase the release of IPEC-J2 cytokine induced by CPB2 toxin, aggravating cell damage.
Experimental example 5 Effect of S100A9 on the CPB2 toxin-induced viability and proliferation of IPEC-J2 cells
1. Experimental methods
(1) IPEC-J2 cell viability induced by S100A9 on CPB2 toxin
The cell suspension was diluted at 1X 10 6 The cells were seeded in 96-well plates and transfected when the cell density reached 70-80% confluence. 24h after transfection, cells were incubated with CPB2 toxin at a concentration of 20. mu.g/mL for 24 h; after the treatment, 10. mu.L of CCK-8 solution (Cell Counting Kit-8 Cell Counting reagent) was added to each well at 37 ℃ with 5% CO 2 And 95% of O 2 Incubate in incubator for 1h, use enzyme-linked immunosorbent assay to determine the absorbance under 450nm wavelength.
(2) Detection of EdU cell Activity of S100A9 on IPEC-J2 induced by CPB2 toxin the number of EDU (5-ethynyl-2' -deoxyuridine ) positive cells was determined by collecting IPEC-J2 cell suspensions at 1X 10 6 Was inoculated on a 24-well plate and cultured for 24 hours. Then use separately
2000 si-NC, si-S100A9, pcDNA3.1, pc-S100A9 were transfected into IPEC-J2 cells for 24 hours. After 24 hours of treatment of IPEC-J2 cells with CPB2 toxin at a concentration of 20. mu.g/mL, the control and CPB2 groups were treated with BeyoClick, respectively TM The EdU-555 cell proliferation assay kit (Biyun day, Shanghai, China) incubated IPEC-J2 cells with EdU (10. mu.M) working solution for 2 hours and stained with Hoechst 33342 for visualization. Detection was performed under a fluorescent inverted microscope (Olympus, japan).
2. Results of the experiment
As shown in FIG. 7, compared with the Control group (Control), the cell viability of the CPB2 group was significantly reduced, and the cell viability of the S100A9 overexpression group (CPB2+ pc-S100A9) was significantly lower than that of the negative Control group (CPB2+ pcDNA3.1). The results for the S100A9 interference group (CPB2+ si-S100A9) are the opposite (FIG. 7A). Cell proliferation was detected by EdU method. The results show that the number of positive cells of the S100A9 overexpression group (CPB2+ pc-S100A9) is very much lower than that of the negative control group (CPB2+ pcDNA3.1). The number of positive cells in the interference group (CPB2+ si-S100A9) was significantly higher than that in the S100A9 interference negative control group (CPB2+ si-NC) (FIG. 7B, FIG. 7C). The above studies indicate that S100a9 inhibits cell viability and cell proliferation.
Experimental example 6 reactive oxygen species ROS and cytotoxic LDH
1. Experimental methods
(1) Reactive Oxygen Species (ROS) level detection
2', 7' -dihydrodichlorofluorescein diacetate (DCFH-DA) is used as a fluorescent probe to detect the level of Reactive Oxygen Species (ROS) in cells. Cells were transfected and seeded, harvested and suspended cells were incubated with 10. mu. mol/L DCFH-DA diluent at 37 ℃ in 5% carbon dioxide for 20 minutes. In order to make the cells completely contact DCFH-DA, every 3-5 minutes reverse mixing, at 1500 rpm centrifugal 5 minutes, discard the supernatant, PBS washing the cell without entering DCFH-DA, with fluorescence enzyme labeling instrument detection of each group of DCFH-DA fluorescence intensity.
(2) Lactate Dehydrogenase (LDH) Activity assay
Referring to the 6-cell culture method of Experimental example 4, after completion of the culture, each group of cells was collected and LDH activity in a sample of IPEC-J2 cells was measured using a lactate dehydrogenase assay kit.
2. Results of the experiment
The fluorescence intensity and Lactate Dehydrogenase (LDH) activity measurement results are shown in FIG. 8, and the results show that compared with the control group, DCFH-DA fluorescence intensity CPB2 toxin treatment IPEC-J2 cells are obviously enhanced (FIG. 8A), and the S100A9 overexpression group (CPB2+ pc-S100A9) is obviously higher than the negative control group (CPB2+ pcDNA3.1); compared with the negative control group (CPB2+ si-NC), the interference group (CPB2+ si-S100A9) has significantly reduced fluorescence intensity. The enzyme activity of Lactate Dehydrogenase (LDH) in the cell culture broth was further detected (fig. 8B); the enzymatic activity of LDH in cell culture fluid was significantly improved after CPB2 toxin treatment. Meanwhile, compared with a transfection overexpression vector control (pcDNA3.1), the transfection S100A9 overexpression vector (pc-S100A9) increases the damage of CPB2 toxin to cells and improves the LDH enzyme activity in a cell culture solution. Whereas, after transfection with the S100a9 inhibitor, the CPB2 toxin was less cytotoxic to the cells.
Experimental example 7 flow cytometry detection of cell cycle
1. Experimental methods
IPEC-J2 cells were digested with 0.25% trypsin, resuspended in 75% pre-chilled ethanol, and incubated overnight at 4 ℃; adding 2 mu L of 10mg/mL RNaseA into the IPEC-J2 cell sample, removing RNA for 30min at 37 ℃, then adding 100 mu L of PI staining solution with the concentration of 100 mu g/mL, and keeping out of the sun for 10 min; finally, IPEC-J2 cell samples were examined with a flow cytometer (CytoFLEX, Beckman, USA) with an excitation wavelength of 488nm and an emission wavelength of 585 ± 21nm, and cell cycle distribution was analyzed with Modfit software.
2. Results of the experiment
The results are shown in FIG. 9, and in FIG. 9, the percentages of the cells at the G0/G1, G2 and S phases in each experimental group are shown, and it can be seen that the percentages of the G0/G1 phases are significantly increased and the S phases are significantly decreased in the pc-S100A9 group, compared with the CPB2 group, while the results are opposite in the si-S100A9 group. Thus, overexpression of S100a9 extended the cell cycle of CPB2 toxin-induced IPEC-J2 cells.
Experimental example 8 expression of Claudin ZO-1, OCLN and CLDN-12
1. Experimental methods
The experimental groups were the same as in example 4, and after completion of the culture, the expression levels of three claudin proteins ZO-1, OCLN and CLDN-12 in each experimental group were examined by RT-qPCR, and the primers are shown in Table 1, and the sequences of the primers are SEQ ID No.13 to SEQ ID No. 18.
2. Results of the experiment
Results as shown in fig. 10, decreased levels of IPEC-J2 cell claudin mRNA expression exposed to CPB2 toxin; compared with the S100A9 overexpression negative control group (CPB2+ pcDNA3.1), the S100A9 overexpression group (CPB2+ pc-S100A9) has the advantages that the expressions of ZO-1, OCLN and CLDN-12 are all obviously reduced. Compared with the S100A9 interference negative control group (CPB2+ si-NC), the S100A9 interference group (CPB2+ si-S100A9) has significantly up-regulated expression of zon-1, OCLN and CLDN-12. The above results indicate that S100A9 disrupts the tight junctions of IPEC-J2 cells induced by CPB2 toxin.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (10)
- The application of an S100A9 inhibitor in preparing a medicine for preventing and treating C-type clostridium perfringens infectious diarrhea.
- 2. The use of claim 1, wherein the inhibitor of S100A9 inhibits the expression of the S100A9 gene in a tissue and/or an intestinal epithelial cell.
- 3. The use according to claim 1, wherein the S100a9 inhibitor reduces the expression of the inflammatory factors IL-6, IL-8, TNF- α and IL-1 β in enterocyte cells under clostridium perfringens type C infection, reducing cell damage.
- 4. The use according to claim 1, wherein the S100a9 inhibitor maintains cell viability of enterocytes under clostridium perfringens type C infection.
- 5. The use according to claim 1, wherein the S100a9 inhibitor increases the proportion of S phase in enterocytes under clostridium perfringens type C infection, maintaining cell proliferation of the enterocytes.
- 6. The use according to claim 1, wherein the S100a9 inhibitor reduces the activity of reactive oxygen species and LDH enzymes in clostridium perfringens type C infected lower intestinal epithelial cells, thereby reducing the cytotoxicity of clostridium perfringens type C against intestinal epithelial cells.
- 7. The use of claim 1, wherein the S100A9 inhibitor maintains significant upregulation of the expression of zonulin ZO-1, OCLN, and CLDN-12, thereby inhibiting decreased epithelial cell tight junction induced by Clostridium perfringens type C infection.
- 8. A medicine containing S100A9 inhibitor for preventing and treating diarrhea is provided.
- 9. The agent for preventing and treating diarrhea according to claim 8, wherein the inhibitor of S100A9 is an RNA interference agent that inhibits transcription of S100A9 gene.
- 10. The drug for preventing and treating diarrhea according to claim 8, wherein the S100A9 inhibitor is a monoclonal antibody or a polyclonal antibody that inhibits S100A9 protein translated from S100A9 gene.
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Citations (6)
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|---|---|---|---|---|
| CN104293788A (en) * | 2014-10-14 | 2015-01-21 | 广西医科大学 | SiRNA inhibiting expression of gene S100A9 and application of siRNA |
| CN111803645A (en) * | 2020-07-24 | 2020-10-23 | 北京大学 | Application of S100A8\ A9 dimer activity inhibitor in prevention and treatment or diagnosis of coronavirus infection |
| CN111840561A (en) * | 2020-08-11 | 2020-10-30 | 大连医科大学附属第一医院 | Application of S100A9 inhibitor in preparation of medicine for treating pancreatitis |
| CN112040982A (en) * | 2018-04-27 | 2020-12-04 | 国立大学法人冈山大学 | anti-S100A 8/A9 antibodies and uses thereof |
| CN113444785A (en) * | 2021-06-28 | 2021-09-28 | 甘肃农业大学 | SSc-miR-122-5p related to piglet C-type clostridium perfringens infectious diarrhea and application thereof |
| WO2021219879A1 (en) * | 2020-04-30 | 2021-11-04 | Aqilion Ab | Treatments of inflammatory bowel disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293788A (en) * | 2014-10-14 | 2015-01-21 | 广西医科大学 | SiRNA inhibiting expression of gene S100A9 and application of siRNA |
| CN112040982A (en) * | 2018-04-27 | 2020-12-04 | 国立大学法人冈山大学 | anti-S100A 8/A9 antibodies and uses thereof |
| WO2021219879A1 (en) * | 2020-04-30 | 2021-11-04 | Aqilion Ab | Treatments of inflammatory bowel disease |
| CN111803645A (en) * | 2020-07-24 | 2020-10-23 | 北京大学 | Application of S100A8\ A9 dimer activity inhibitor in prevention and treatment or diagnosis of coronavirus infection |
| CN111840561A (en) * | 2020-08-11 | 2020-10-30 | 大连医科大学附属第一医院 | Application of S100A9 inhibitor in preparation of medicine for treating pancreatitis |
| CN113444785A (en) * | 2021-06-28 | 2021-09-28 | 甘肃农业大学 | SSc-miR-122-5p related to piglet C-type clostridium perfringens infectious diarrhea and application thereof |
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