CN113440505A - Application of N-acetylcysteine in preparation of medicine for treating pig intestinal injury caused by glyphosate - Google Patents
Application of N-acetylcysteine in preparation of medicine for treating pig intestinal injury caused by glyphosate Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
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Abstract
The invention discloses application of N-acetylcysteine in preparing a medicament for treating pig intestinal injury caused by glyphosate, which can effectively relieve or treat the problem of medicaments for treating intestinal cell injury induced by glyphosate. The application comprises relieving activity inhibition in porcine intestinal epithelial cells caused by glyphosate, and reducing active oxygen content in the porcine intestinal epithelial cells induced by the glyphosate and the relative expression quantity of mRNA of interleukin IL-6. The method has the advantages that the expression of the conversion ratio of the swine intestinal epithelial cell microtubule-associated protein light chain 3(LC3) from LC3-I to LC3-II induced by glyphosate is down-regulated, and the protein consumption of autophagy-related substrate P62 in the swine intestinal epithelial cells caused by the glyphosate is up-regulated, so that the method has the advantage of protecting the integrity and health of animal intestines.
Description
Technical Field
The invention relates to the field of veterinarians, in particular to application of N-acetylcysteine in preparing a medicine for treating pig intestinal injury caused by glyphosate.
Background
N-acetylcysteine, hereinafter referred to as NAC, has the following structural formula:
NAC is a sulfhydryl-containing amino acid derivative, which can be used as a Reactive Oxygen Species (ROS) scavenger and a substrate of glutathione to resist tissue oxidative damage caused by different reasons, and is a widely-used drug with research value. Mainly as an antioxidant, active oxygen (ROS), an electrophilic metabolite of acetaminophen in vivo, such as hydroxyl (OH), nitrogen dioxide (NO2) and hydrogen peroxide (H)2O2) They are neutralized by a rapid reaction. In addition, NAC regulates the metabolic activity of cells, regulates gene expression and signal transduction systems, and has been widely used in clinical and experimental applications.
Glyphosate (GLP) is a systemic and efficient broad-spectrum herbicide with organophosphorus properties, is the herbicide with the largest use amount, the widest application and the highest annual sale amount worldwide, and the use amount is continuously increased. Glyphosate is widely present in soil and surface water as a contaminant, and animal feed also contains glyphosate residues of varying concentrations. Human beings inevitably contact glyphosate by various means such as water, food, air and the like. Glyphosate can cause damage to a plurality of organs of animals, and when the animals are exposed to glyphosate for a long time, endocrine system, immune system, nervous system, digestive system, cells and genes can be damaged.
The pig intestinal tract is an organ integrating functions of digestion, absorption, internal secretion, immunity and defense, and is easily damaged by harmful exogenous substances such as pesticides, toxins and the like. Porcine intestinal epithelial cells (IPEC-J2) are the first physical barrier against external stimuli. IPEC-J2 was derived from non-suckling piglet jejunal epithelial cells. It is a non-transformed, non-tumorigenic intestinal epithelial cell line, with comparable activity in vivo. Is suitable for in vitro models and can represent the porcine intestinal epithelial cells under normal physiological conditions. In conclusion, how to develop a drug capable of alleviating or treating porcine intestinal injury induced by glyphosate is a technical problem to be solved urgently.
Disclosure of Invention
Based on the defects, the invention provides the application of N-acetylcysteine in preparing the medicine for treating the pig intestinal injury caused by glyphosate, so as to solve the problem that the medicine for effectively relieving or treating the intestinal cell injury induced by the glyphosate is lacked in the prior art.
The technical scheme of the invention is as follows: application of N-acetylcysteine in preparing medicine for treating pig intestinal tract injury caused by glyphosate is disclosed.
Further, the use as described above for alleviating inhibition of activity in porcine intestinal epithelial cells caused by glyphosate.
Further, the application is used for reducing the active oxygen content in the porcine intestinal epithelial cells caused by the glyphosate.
Further, the application is used for reducing the expression quantity of mRNA (messenger ribonucleic acid) of the proinflammatory factor IL-6 in the porcine intestinal epithelial cells caused by the glyphosate.
Further, the application is used for down regulating the expression of autophagy-related protein LC3-II/LC3-I ratio in porcine intestinal epithelial cells caused by glyphosate.
Further, the use as described above for up-regulating protein expression of autophagy-related substrate P62 in porcine intestinal epithelial cells inhibited by glyphosate.
The medicine prepared from the N-acetylcysteine is tablets, capsules, granules, dripping pills, suspensions, syrups, various enteric preparations or injections. The various dosage forms can be prepared according to conventional manufacturing methods in the pharmaceutical art, for example by mixing the active ingredient with one or more carriers and then formulating it into the desired dosage form.
The invention has the following advantages and beneficial effects: n-acetylcysteine can relieve glyphosate-induced IPEC-J2 cell proliferation toxicity, oxidation, inflammation and autophagy, and has the advantages of protecting animal intestinal tract integrity and health.
Drawings
FIG. 1 is a graph of the effect of NAC on the IPEC-J2 cell viability damage caused by GLP protected by NAC provided by an embodiment of the present invention, marked differently with lower case letters indicating significant differences and the same letters indicating insignificant differences.
FIG. 2 is a graph of fluorescence of cellular reactive oxygen species induced by NAC inhibition GLP provided by an embodiment of the invention.
FIG. 3 is a graph of the effect of NAC on the inhibition of GLP-induced reactive oxygen species in cells provided by an embodiment of the invention, marked with different lower case letters for significant difference and the same letters for insignificant difference.
FIG. 4 is a graph showing the effect of NAC on the inhibition of the relative expression level of the GLP-induced cytokine IL-6mRNA, which is provided by the embodiment of the present invention and marked with different lower case letters to show significant difference, and the same letter to show insignificant difference.
Fig. 5 is a western blot of NAC effect on GLP-induced autophagy-related proteins provided by an embodiment of the invention.
Fig. 6 is a western blot quantification of NAC-mitigating GLP-induced consumption of autophagy-related protein P62 provided by an embodiment of the invention. The notation of different lower case letters indicates significant differences, and the same letter indicates insignificant differences.
Fig. 7 is a western blot quantification map of NAC inhibition GLP induced lipidation of autophagy-related protein LC3 provided by an embodiment of the invention.
The notation of different lower case letters indicates significant differences, and the same letter indicates insignificant differences.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials: phosphate Buffered Saline (PBS), 0.25% pancreatin-ethylenediaminetetraacetic acid (EDTA), penicillin-streptomycin, dimethyl sulfoxide (DMSO) and thiazolyl tetrazolium bromide (MTT), active oxygen assay kits were purchased from Biyuntian biology, China; NAC purity ≥ 99% was purchased from Sigma-Aldrich (USA); DMEM/F12 medium and Fetal Bovine Serum (FBS) were purchased from Hyclone (USA). Glyphosate (Noda) was purchased from Monsanto Inc. (USA).
Practice of the inventionIn the example, the IPEC-J2 cell line is a gift from the animal science and technology institute of Chinese university of agriculture. The IPEC-J2 cell culture solution is DMEM/F12 culture medium containing 10% FBS and 1% penicillin-streptomycin, and is inoculated to 25cm2Placing in a culture flask at 37 deg.C and 5% CO2Culturing in an incubator with the concentration. NAC and glyphosate are diluted into different concentrations by culture solution without serum and antibiotics, and are prepared for use.
In the following examples, three independent experiments were performed for each treatment in the experiment. The experimental data were collated and calculated using Microsoft Excel2016, and then subjected to One-Way analysis of variance (One-Way ANOVA) using SPSS 19.0 statistical software (Chicago, USA), with multiple comparisons using the Duncan method. The test results are expressed as Mean (Mean) and Standard Error (SEM). P <0.05 was used as a criterion for significance of difference.
Example 1 NAC alleviation of GLP-induced IPEC-J2 cell proliferative toxicity
IPEC-J2 cells were co-cultured with different concentrations of NAC (0, 0.5, 1mM) and different concentrations of GLP (0, 30, 60mg/L) for 12h, with the results shown in FIG. 1, where NAC at different concentrations reduced cell damage caused by GLP. Cell viability was significantly improved when cells were treated with 1mM NAC (P < 0.05). Based on the above results, 1mM NAC incubated cells were selected for 12h for subsequent experiments.
Example 2 NAC Mitigation of GLP-induced IPEC-J2 cell injury
NAC inhibits GLP-induced IPEC-J2 cell active oxygen content
IPEC-J2 cells in logarithmic growth phase were collected, digested with pancreatin, and cultured at 2.5X 105cell/well density cells were cultured in 6-well plates until the cell fusion rate reached 80%, then cells were treated in four different ways (control, GLP, NAC, GLP + NAC), followed by 1: 1000 with serum-free medium diluted DCFH-DA, to a final concentration of 10 u mol/L. After the treatment time was complete, the cell culture fluid was removed and washed twice with PBS. Diluted DCFH-DA was added. Incubate at 37 ℃ for 20min in a cell culture box. Cells were washed three times with PBS to sufficiently remove DCFH-DA that did not enter the cells. Using 488nm excitation wavelength and 525nm emission wavelength by a fluorescence microplate reader, detecting fluorescence before and after stimulation in real timeThe intensity of the light. And observed under a fluorescent microscope.
As can be seen in fig. 2-3, the addition of NAC significantly reduced the active oxygen content of IPEC-J2 cells (P <0.05), thus preliminarily concluding that NAC can alleviate GLP-induced oxidative damage.
NAC alleviation of GLP-induced relative mRNA expression level of IPEC-J2 cytokine IL-6
The RNA concentration is detected by adopting a TRIZOL total RNA extraction method and ultraviolet light absorption, and the ratio of A260/A280 is between 1.8 and 2.0. And the integrity of the RNA was verified by agarose gel. Total RNA was reverse transcribed into cDNA using SYBR Premix Ex TaqTM kit according to the manufacturer's instructions and used for real-time polymerase chain reaction. And carrying out Real-Time PCR by using a SYBR Real-Time PCR kit Real-Time PCR detection system. The PCR conditions were pre-denaturation: 95 ℃, 30s, cycle 1, RT-PCR reaction: the cycle was repeated 40 times at 95 ℃ for 5 seconds, 61 ℃ for 34 seconds. Beta-actin was used as an internal reference gene, and the relative expression level of each gene was analyzed by the 2-. DELTA.CT method. The primers used in the fluorescent quantitative PCR were designed and synthesized by Biotechnology engineering Co., Ltd (Shanghai) according to the known gene sequences in Genbank, and the IL-6 primer sequences were as follows:
F:AATGTCGAGGCCGTGCAGATTAG，
R:TTCATCCACTCGTTCTGTGACTGC。
as can be seen in fig. 4, the addition of NAC significantly reduced mRNA expression of IL-6 by IPEC-J2 cells (P <0.05), thus preliminarily inferring that NAC can alleviate GLP-induced inflammatory injury.
NAC alleviation of GLP-induced levels of IPEC-J2 autophagy
The sample lysate was centrifuged at 12000rPm, 4 ℃ for 10min, and then the protein was quantitatively processed. 40 μ g of total protein was electrophoresed through SDS polyacrylamide gel, followed by transferring the protein onto PVDF membrane by wet electric transfer method. The working solutions of the LC3 and p62 antibodies were placed in a sealed PVDF membrane, and incubated overnight at four degrees, followed by incubation with a secondary antibody against goat IgG-HRP. Protein signals are detected using an enhanced chemiluminescence detection system. The films were scanned and the optical density values of the target bands were analyzed using a Gel image processing system (Gel-Pro-Analyzer software), as shown in fig. 5, a western blot of NAC effects on GLP-induced autophagy-related proteins.
As can be seen from fig. 6, the addition of NAC significantly increased P62 protein expression (P <0.05), and as can be seen from fig. 7, the addition of NAC significantly decreased the LC3-II/LC3-I protein ratio (P <0.05) of IPEC-J2 cells. It was preliminarily concluded that NAC can alleviate GLP-induced autophagy.
Claims (6)
1. Application of N-acetylcysteine in preparing medicine for treating pig intestinal tract injury caused by glyphosate is disclosed.
2. Use according to claim 1, characterized in that: use for alleviating inhibition of activity in porcine intestinal epithelial cells caused by glyphosate.
3. Use according to claim 1, characterized in that: is used for reducing the active oxygen content in the porcine intestinal epithelial cells caused by the glyphosate.
4. Use according to claim 1, characterized in that: is used for reducing the expression quantity of mRNA of proinflammatory factor IL-6 in porcine intestinal epithelial cells caused by glyphosate.
5. Use according to claim 1, characterized in that: used for down regulating the expression of autophagy-related protein LC3-II/LC3-I ratio in porcine intestinal epithelial cells caused by glyphosate.
6. Use according to claim 1, characterized in that: used for up-regulating the protein expression of autophagy-related substrate P62 in porcine intestinal epithelial cells inhibited by glyphosate.
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