Application of 3, 4-dihydroxybenzylidene acetone in preparation of medicine for relieving intestine injury of Leizhou black ducks

Abstract

The invention discloses application of 3, 4-dihydroxybenzylidene acetone (DBL) in preparation of a medicine for relieving intestine injury of a Leizhou black duck, and belongs to the technical field of biological medicine. According to the application disclosed by the invention, the DBL can be used for improving the antioxidant enzyme activity, clearing ROS (reactive oxygen species), recovering mitochondrial membrane potential, relieving mitochondrial dysfunction, promoting mitochondrial autophagy and clearing damaged mitochondria by mediating cGAS / STING and Parkin / PINK1 signal channels; in addition, by mediating a Bax / Bcl-2 signal channel, the expression of Bcl-2 can be improved, and the expression of Caspase-3 and Caspase-9 can be reduced, so that the apoptosis can be relieved. The invention discloses an action mechanism of DBL for guaranteeing cell metabolism, relieving inflammatory response and relieving lipopolysaccharide-induced Leizhou black duck intestinal injury through double targets, and provides a candidate medicine for preventing and treating intestinal diseases caused by gram-negative bacteria of livestock.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of 3,4-dihydroxybenzalacetone in the preparation of a drug for alleviating intestinal injury of Leizhou black ducks. Background Art

[0002] Currently, antibiotics are considered the first choice for treating bacterial diseases. However, microbial resistance caused by antibiotics and antibiotic residues have severely affected the quality and safety of livestock and poultry eggs and meat. Therefore, the livestock and poultry breeding industry urgently needs safe and efficient antibiotic alternatives. Thus, it is particularly important to find natural active substances as antibiotic alternatives.

[0003] Bacterial enteritis and intestinal injury caused by the infection of Gram-negative bacteria such as Escherichia coli have severely affected the healthy development of the livestock and poultry breeding industry. The endotoxin of Gram-negative bacteria is the main cause of intestinal injury, and lipopolysaccharide (LPS) is the main toxic component of endotoxin in the outer membrane of Gram-negative bacteria. It can cause the body's immune response and activate monocytes and macrophages, leading to the activation of intracellular signaling molecules and the release of inflammatory factors, causing the cells to have inflammatory and oxidative stress responses. The initial organ damaged by LPS is the intestinal tissue. After LPS enters the intestine, it increases the intestinal barrier permeability, damages the mucus layer, causes apoptosis of intestinal epithelial cells and mitochondrial dysfunction, etc., and then leads to the formation of intestinal inflammation and ulcers, causing intestinal injury, and further affecting the growth and development and immune function of animals, resulting in disease outbreaks and affecting the economic benefits of the livestock and poultry breeding industry.

[0004] Currently, there have been reports on natural active substances that can be used to treat enteritis. For example, resveratrol can play an immunomodulatory role and prevent the occurrence of colitis by regulating the expression of inflammatory factors and reducing the differentiation of T cells in the colon. However, the effect of resveratrol in alleviating LPS-induced intestinal injury is limited, and it is necessary to continuously develop natural active substances that can be used to prevent and treat LPS-induced intestinal injury. 3,4-Dihydroxybenzalacetone (DBL) is a polyphenolic compound with various biological activities, including anti-inflammatory, antioxidant, anti-tumor, immunomodulatory, etc. The prevention and treatment of livestock and poultry intestinal injury caused by Gram-negative bacteria is a complex process that requires the synergistic action of multiple pathways, that is, not all drugs with anti-inflammatory effects can necessarily alleviate LPS-induced intestinal injury. Currently, there is no report on the use of DBL to alleviate LPS-induced intestinal injury in Leizhou black ducks. Summary of the Invention

[0005] The object of the present invention is to provide the application of 3,4-dihydroxybenzalacetone in the preparation of a drug for alleviating intestinal injury of Leizhou black ducks, so as to solve the problems existing in the above prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the application of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for relieving intestinal injury of Leizhou black ducks.

[0008] Another technical solution of the present invention is the application of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for relieving mitochondrial dysfunction in the intestine of Leizhou black ducks.

[0009] A third technical solution of the present invention is the application of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for inhibiting apoptosis of intestinal cells of Leizhou black ducks.

[0010] Based on the above technical solutions, the present invention has the following technical effects:

[0011] The present invention discovers that 3,4-dihydroxybenzylideneacetone can restore the mitochondrial membrane potential, relieve mitochondrial dysfunction, promote mitophagy and remove damaged mitochondria by mediating the cGAS / STING and Parkin / PINK1 signaling pathways; it can also mediate the Bax / Bcl-2 signaling pathway, increase the expression of Bcl-2, and decrease the expression of Caspase-3 and Caspase-9, thereby relieving cell apoptosis. The present invention elaborates on the mechanism of action of 3,4-dihydroxybenzylideneacetone in protecting cell metabolism through dual targets, scavenging ROS, reducing inflammatory responses, and relieving lipopolysaccharide-induced intestinal injury in Leizhou black ducks, providing a candidate drug for the prevention and treatment of intestinal diseases caused by Gram-negative bacteria in livestock and poultry, contributing to the development of natural drugs that can effectively treat intestinal injuries, and contributing to the development of the livestock and poultry breeding industry in China. Description of the Drawings

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0013] Figure 1 Shows the effect of DBL on the intestinal injury model of Leizhou black ducks. Among them, A is a schematic diagram of the grouping design of Leizhou black ducks, and B is a representative microscopic image (100×) of H&E-stained small intestine sections.

[0014] Figure 2Effect of DBL on the intestinal barrier integrity of Leizhou black ducks. Among them, A is the activity level of DAO; B is the detection result of immunoblot analysis of ZO-1, occludin, and Claudin1 proteins, and C is the relative expression level of ZO-1, Occludin, and Claudin1 proteins.

[0015] Figure 3 Effect of DBL on inflammatory factors in the serum of Leizhou black ducks induced by LPS. Among them, A is the detection result of IL-18, IL-1β, IL-6, and TNF-α levels, and B is the detection result of NO level.

[0016] Figure 4 Effect of DBL on oxidative stress in the serum of Leizhou black ducks induced by LPS. Among them, A is the detection result of SOD, B is the detection result of GSH-Px, C is the detection result of MDA, and D is the detection result of ROS.

[0017] Figure 5 Effect of DBL on the mitochondrial membrane potential of the intestinal tissue of Leizhou black ducks induced by LPS (200×).

[0018] Figure 6 Effect of DBL on the mitochondrial dysfunction signaling pathway in the intestinal injury of Leizhou black ducks induced by LPS. Among them, A is the mRNA levels of cGAS and STING1, B is the immunoblot analysis results of TBK1, p-TBK1, IRF3, cGAS, and STING1 proteins, and C is the relative expression levels of p-TBK1, IRF3, cGAS, and STING1 proteins.

[0019] Figure 7 Effect of DBL on the mitophagy signaling pathway in the intestinal injury of Leizhou black ducks induced by LPS. Among them, A is the mRNA levels of p62 and NLRP3, B is the mRNA levels of Parkin, ATG5, PINK1, and LC3, C is the immunoblot analysis results of p62, NLRP3, Parkin, ATG5, PINK1, LC3, and ATG7 proteins, D is the relative expression levels of p62, NLRP3, Parkin, and ATG5 proteins, and E is the relative expression levels of PINK1, LC3, and ATG7 proteins.

[0020] Figure 8 Effect of DBL on the apoptosis rate of intestinal tissue cells of Leizhou black ducks induced by LPS. Among them, A is the apoptosis detection result, and B is the apoptosis data analysis.

[0021] Figure 9To investigate the effect of DBL on the cell apoptosis signaling pathway in LPS-induced intestinal injury in Leizhou black ducks. Among them, A shows the mRNA levels of BAK1, Caspase-3, Caspase-9, and CytC; B shows the results of Western blot analysis of the proteins of BAK1, Caspase-3, Caspase-9, CytC, BAX, and BCL-2; C shows the relative expression levels of BAK1, Caspase-3, and Caspase-9 proteins; D shows the relative expression levels of CytC, BAX, and BCL-2 proteins.

[0022] Figure 10 Schematic diagram of the mechanism of DBL on LPS-induced intestinal injury in Leizhou black ducks.

[0023] Note: "*" indicates compared with the control group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; "#" indicates compared with the LPS group, # indicates P < 0.05, ## indicates P < 0.01, indicates P < 0.001. Detailed implementation manners

[0024] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0025] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of this application are only exemplary.

[0028] The terms "comprising", "including", "having", "containing", etc. used in this text are all open-ended terms, meaning including but not limited to.

[0029] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the art. Unless otherwise specified, the reagents or raw materials used are all purchased from commercial channels or are publicly available.

[0030] The LPS used in the examples of the present invention was purchased from Sigma, with the product number L2880.

[0031] All data in the present invention are expressed as mean ± standard deviation (x±s). Graphad Prism software was used for drawing, and SPSS 23.0 software was used for statistical analysis; significant differences are expressed as: compared with the control group, "*" is used, *(P<0.05), **(P<0.01), ***(P<0.001); compared with the LPS group, "#" is used, #(P<0.05), ##(P<0.01), (P<0.001).

[0032] The examples of the present invention provide the application of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for relieving intestinal injury in Leizhou black ducks.

[0033] In some specific embodiments, the intestinal injury is intestinal injury caused by lipopolysaccharide.

[0034] In some specific embodiments, the 3,4-dihydroxybenzylideneacetone effectively maintains the integrity of the intestinal barrier and relieves lipopolysaccharide-induced intestinal pathological injury in Leizhou black ducks by reducing inflammatory factors and upregulating the expression of intestinal tight junction proteins.

[0035] The examples of the present invention also provide the application of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for relieving intestinal mitochondrial dysfunction in Leizhou black ducks.

[0036] In some specific embodiments, the mitochondrial dysfunction is intestinal mitochondrial dysfunction caused by lipopolysaccharide.

[0037] In some specific embodiments, the 3,4-dihydroxybenzylideneacetone relieves lipopolysaccharide-induced intestinal mitochondrial dysfunction in Leizhou black ducks by mediating the cGAS / STING and Parkin / PINK1 signaling pathways, increasing the activity of antioxidant enzymes, scavenging ROS, restoring mitochondrial membrane potential, promoting mitophagy and clearing damaged mitochondria.

[0038] The examples of the present invention also provide the application of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for inhibiting apoptosis of intestinal cells in Leizhou black ducks.

[0039] In some specific embodiments, the intestinal apoptosis is intestinal apoptosis caused by lipopolysaccharide.

[0040] In some specific embodiments, 3,4-dihydroxybenzylideneacetone inhibits lipopolysaccharide-induced intestinal apoptosis in Leizhou black ducks by regulating the Bax / Bcl-2 signaling pathway, increasing Bcl-2 expression, and decreasing Caspase-3 and Caspase-9 expression.

[0041] Example 1 DBL alleviates LPS-induced intestinal injury in Leizhou black ducks

[0042] 1. Experimental animals and grouping design

[0043] Forty-two 1-day-old Leizhou black ducks were taken, adaptively fed for 7 days, and randomly divided into 6 groups with 7 ducks in each group, namely:

[0044] Control group: Fed with saline-mixed feed for 7 days and then intraperitoneally injected with saline continuously for 3 days;

[0045] LPS group: Fed with saline-mixed feed for 7 days and then intraperitoneally injected with 10 mg / kg LPS continuously for 3 days;

[0046] LPS + DBL low concentration (0.01% volume fraction), LPS + DBL high concentration (0.04% volume fraction), and LPS + resveratrol (RES) (0.04% volume fraction) groups: Each group was fed with DBL and RES for 7 days and then intraperitoneally injected with LPS continuously for 3 days;

[0047] DBL (0.04% volume fraction) group: Fed with DBL for 7 days and then intraperitoneally injected with saline continuously for 3 days.

[0048] After the last administration, only fast, drink water normally, collect blood from the heart into a 15 mL anticoagulation tube after 12 h, centrifuge at 3000 rpm for 10 min, and take the supernatant. Collect small intestine tissue samples, fix part of the small intestine tissue in 4% paraformaldehyde for preparing pathological sections. Preserve part of the small intestine tissue at -80 °C for subsequent detection. Feed the same feed during the experiment and drink water freely.

[0049] 2. Histopathological observation of the intestine

[0050] The intestinal tissue samples of Leizhou black ducks in each group were made into paraffin sections and subjected to pathological observation by HE (hematoxylin-eosin staining method) to observe the effect of DBL on LPS-induced intestinal injury in Leizhou black ducks. The results of the effect of DBL on the intestinal tissue of Leizhou black ducks with LPS-induced intestinal injury are as follows Figure 1As shown. Compared with the control group, the intestinal wall of the LPS group became thinner, the intestinal villi were significantly broken, and intestinal mucosal inflammation was accompanied. Compared with the LPS group, the DBL and RES intervention groups could significantly relieve the damage of the morphological structure of the intestinal villi. When the DBL concentration was 0.04%, the preventive effect was the best, the intestinal wall thickened significantly, the intestinal villi became longer, and the shedding decreased. This indicates that DBL can significantly improve the pathological changes of duck intestinal tissues induced by LPS.

[0051] 3. Detection of DAO (diamine oxidase) activity

[0052] DAO is an important indicator for detecting intestinal injury. By detecting the DAO activity in intestinal tissues, the protective effect of DBL on the small intestine can be evaluated. The higher the activity, the better the protective effect on the small intestine. Take the collected small intestine tissues of Leizhou black ducks in each group, and use a tissue homogenizer to fully grind the small intestine tissues at 4°C, 65 Hz, and 120 s. Centrifuge at 8000 rpm and 4°C for 10 min, and take the supernatant; the DAO activity is operated according to the instructions of the DAO kit (Beijing Solarbio Science & Technology Co., Ltd.). The results of the effect of DBL on the DAO activity in the small intestine tissues of Leizhou black ducks with LPS-induced intestinal injury are as Figure 2 shown in Figure A. Compared with the control group, the serum DAO activity was significantly increased under the action of LPS (P<0.001). Pretreatment with DBL could significantly reduce the serum DAO activity (P<0.001). When the dose of DBL was 0.04%, the DAO activity in the serum was the lowest and lower than that of 0.04% RES. The above results indicate that DBL can significantly increase the DAO activity in the small intestine of Leizhou black ducks induced by LPS, thereby alleviating the intestinal injury of Leizhou black ducks induced by LPS.

[0053] 4. Protein immunoblot analysis (WB) to detect intestinal tight junction proteins

[0054] In the present invention, the conventional protein immunoblot analysis method was used to analyze the expression levels of intestinal tight junction proteins ZO-1, Occludin, and Claudin1, and to observe the effect of DBL on the tight junction proteins of Leizhou black ducks with intestinal injury. The results are as Figure 2 shown in Figures B-C. Compared with the control group, the expression of tight junction proteins in the LPS group was significantly decreased (P<0.001). Compared with the LPS group, the DBL and RES intervention groups could significantly increase the expression levels of ZO-1, Occludin, and Claudin1 (P<0.05). When treated with 0.04% DBL, the expression of intestinal tight junction proteins was not significantly different from that of the control group (P>0.05). The above results suggest that DBL can regulate the expression of tight junction proteins, thereby protecting the intestinal barrier.

[0055] 5. Detection of inflammatory factors

[0056] Operate according to the instructions of the ELISA kit (Beijing Qisong Biotechnology Co., Ltd.) to detect the contents of IL-18, IL-1β, IL-6, TNF-α, and NO in the serum of Leizhou black ducks in each treatment group. The results are as Figure 3 shown. Compared with the control group, the expressions of IL-18, IL-1β, IL-6, TNF-α, and NO in the serum of Leizhou black ducks in the LPS group were significantly increased (P<0.001); compared with the LPS group, 0.01% and 0.04% DBL both decreased the contents of inflammatory factors and NO. After treatment with 0.04% DBL, there was no significant difference in the levels of IL-18, IL-1β, IL-6, TNF-α, and NO compared with the control group (P>0.05). The above results indicate that DBL can effectively inhibit the expression of inflammatory factors in the small intestine tissue of Leizhou black ducks.

[0057] To explore whether DBL can protect against LPS-induced intestinal injury and further investigate its protective mechanism, an intestinal injury model of Leizhou black ducks was constructed. Histopathological results showed that 0.04% DBL could significantly improve the intestinal villus breakage, abnormal changes in crypt structure, and inflammatory cell infiltration in the LPS-induced intestinal injury model. ELISA test results proved that 0.04% DBL could significantly reduce the expressions of inflammatory factors IL-6, TNF-α, IL-18, IL-1β, and NO in the serum, and the effect was better than that of RES at the same concentration. The present invention shows that DBL can reduce the increase in the contents of IL-6, TNF-α, and IL-1β induced by LPS in mice. DAO, as a highly active intracellular enzyme unique to intestinal villi, can affect cell repair and intestinal mucosal barrier by regulating the intracellular environment. When the intestine is damaged, DAO will be released into the intestinal lumen and absorbed into the blood through capillaries, resulting in an increase in the activity of DAO in the serum. Therefore, DAO is an ideal indicator of the structural integrity of the intestinal mucosa. Tight junction proteins mainly include Occludin, Claudins, and ZO-1. In this study, the detection results of DAO activity and tight junction proteins confirmed that 0.04% DBL could restore the intestinal injury caused by LPS.

[0058] Example 2 DBL alleviates LPS-induced mitochondrial dysfunction in Leizhou black ducks

[0059] 1. Experimental animals and grouping design

[0060] The same method and grouping design as in Example 1 were used.

[0061] 2. Detection of oxidation indexes

[0062] According to the instructions of the SOD commercial kit (Nanjing Jiancheng Bioengineering Institute) and the MDA and GSH-Px activity detection kit (Beijing Solebow Technology Co., Ltd.), the contents of SOD, MDA, and GSH-Px in the serum of Leizhou black ducks in each treatment group were detected. According to the instructions of the ROS determination kit (Nanjing Jiancheng Bioengineering Institute), the ROS content in the intestinal tissue of Leizhou black ducks was detected. DCFH-DA solution was added to the treated intestinal tissue suspension, the final concentration was adjusted to 20 μmol / L, the probe was loaded at 37°C for 40 minutes, and the ROS content was detected by a fluorescent microplate reader. (Excitation wavelength 488nm, emission wavelength 525nm).

[0063] The results are as follows Figure 4 As shown in the results, compared with the control group, LPS significantly reduced the activities of SOD and GSH-Px in serum and increased the content of MDA (P<0.001); compared with the LPS group, DBL pretreatment could increase the content of SOD and GSH-Px in intestinal tissue and reduce the content of MDA (P<0.001). The results of ROS fluorescence intensity showed that compared with the control group, the ROS fluorescence intensity in the LPS group was significantly increased (P<0.001); DBL could significantly reduce the accumulation of ROS caused by LPS (P<0.001) and reduce the fluorescence intensity, among which the ROS fluorescence intensity in the LPS+DBL (0.04%) group had no significant difference with that in the CON group (P>0.05). This indicates that DBL can inhibit the oxidative stress in the small intestinal tissue induced by LPS.

[0064] 3. Mitochondrial membrane potential detection

[0065] Mitochondrial membrane potential (MMP) is a key factor in maintaining the normal function of mitochondria and is closely related to the metabolic activity of normal cells. The decrease of MMP is a landmark event in the early stage of cell apoptosis. In order to explore the effect of DBL on LPS-induced changes in mitochondrial membrane potential of intestinal tissue of Leizhou black duck, the JC-1 staining method was used to evaluate the mitochondrial membrane potential. 30 mg of each treated intestinal tissue was weighed, crushed and digested, filtered and resuspended, and JC-1 staining working solution was added in a 1:1 ratio. The cells were incubated at 37°C for 20 minutes, centrifuged at 600g for 4 minutes, and the supernatant was discarded. The cells were washed three times with JC-I staining buffer (1×) and resuspended, and observed under a fluorescence microscope (excitation wavelength 515nm, emission wavelength 585nm). The results are as follows Figure 5 As shown in the figure, compared with the control group, the green fluorescence in the LPS group was enhanced and the mitochondrial membrane potential was significantly reduced. Compared with the LPS group, DBL intervention can enhance red fluorescence and reduce green fluorescence, which is manifested as an increase in mitochondrial membrane potential. This indicates that DBL can restore the LPS-induced decrease in mitochondrial membrane potential in small intestinal tissue, restore mitochondrial function, and have a protective effect on mitochondria.

[0066] 4. Detection by quantitative RT-PCR analysis (qRT-PCR) and Western blotting analysis (WB)

[0067] In the present invention, RNA of the small intestine tissue of Leizhou black ducks was extracted using TRIzol, and cDNA was obtained by reverse transcribing the RNA according to the RNA reverse transcription kit. SYRB Green was used for quantitative RT-PCR analysis, and the results were calculated by the 2 -ΔΔCT method and normalized to the β-actin expression level. The expression levels of the cGAS / STING signaling pathway and the Parkin / PINK1 signaling pathway were detected by the same Western blotting method as in Example 1 to observe the effect of DBL on the cGAS / STING signaling pathway and the Parkin / PINK1 signaling pathway in Leizhou black ducks with intestinal injury. The proteins to be detected included TBK1, p-TBK1, IRF3, cGAS, STING1, p62, NLRP3, Parkin, ATG5, PINK1, LC3, and ATG7.

[0068] The primer sequences used for quantitative RT-PCR analysis in the present invention are shown in Table 1:

[0069] Table 1 Primer sequences used for quantitative RT-PCR analysis

[0070]

[0071]

[0072] The results are as Figure 6 and 7As shown, compared with the control group, LPS significantly increased the mRNA levels of cGAS, STING1, p62, and NLRP3 (P<0.001), and significantly decreased the mRNA levels of Parkin, ATG5, PINK1, and LC3 (P<0.001); compared with the LPS group, DBL could significantly change this trend, and the treatment effect of 0.04% DBL was better than that of RES at the same concentration. The WB results showed that compared with the control group, the phosphorylation level of TBK1, the protein expressions of IRF3, cGAS, STING1, p62, and NLRP3 in the LPS group were significantly increased (P<0.001), and the protein expressions of Parkin, ATG5, PINK1, LC3, and ATG7 were significantly decreased (P<0.001); compared with the LPS group, different concentrations of DBL decreased the phosphorylation level of TBK1, the protein expressions of IRF3, cGAS, and STING1, promoted the increased expression of mitophagy proteins, decreased the protein content of p62 and the protein expression of the inflammasome NLRP3. After treatment with 0.04% DBL, there was no significant difference in protein expression compared with the control group (P>0.05), and the effect was better than that of RES at the same concentration. This indicates that DBL can regulate the mitochondrial dysfunction signaling pathway, protect mitochondrial function, activate the mitophagy pathway in the intestine, and inhibit LPS-induced mitochondrial dysfunction.

[0073] When LPS induces mitochondrial damage, the intracellular redox balance will be disrupted, manifested as the accumulation of peroxidation products and the inhibition of the expression and activity of antioxidant enzymes. SOD and GSH-px are important enzymes for scavenging free radicals and resisting oxidative damage in the intestine. SOD can convert superoxide anion radicals into H2O2, block the excessive accumulation of free radicals, prevent lipid peroxidation of cell membranes, and thus reduce the production of MDA. GSH-px can further decompose H2O2 into non-toxic H2O and O2 to complete the thorough scavenging of free radicals. The two can cooperate with each other to jointly maintain the intracellular redox balance. In this study, the determination of mitochondrial-related indicators, the activities of SOD and GSH-px, and the contents of ROS and MDA were consistent with the in vitro detection results. 0.04% DBL could also increase the activities of antioxidant enzymes and significantly reduce the contents of ROS and MDA in the intestinal injury model. The present invention found that 0.04% DBL could inhibit the phosphorylation level of TBK1, the expression of IRF3 protein, and the mRNA and protein expressions of cGAS and STING1 in the intestinal injury model of Leizhou black ducks, and the effect of DBL was better than that of RES at the same concentration, indicating that DBL could prevent and treat LPS-induced intestinal inflammatory injury by improving mitochondrial dysfunction. Mitophagy is an extremely complex physiological process that maintains the balance of mitochondrial mass and quantity to stabilize the intracellular environment. The NLRP3 inflammasome is a complex composed of multiple proteins and can be divided into three parts: the sensor NLRP3, the adaptor protein ASC, and the effector enzyme pro-caspase-1. The interaction between these proteins constitutes the regulatory network of mitophagy to ensure the effective clearance of damaged mitochondria and the maintenance of cell function. In the present invention, the results of RT-qPCR and Western blot showed that the treatment with 0.04% DBL could promote the expression levels of PINK1, Parkin, ATG5, ATG7, and LC3II / I in intestinal tissues, and reduce the contents of p62 and NLRP3, suggesting that DBL could promote the specific binding of p62 and NLRP3, reduce the accumulation of NLRP3, and at the same time consume p62 to increase the formation of autophagosomes by enhancing the mitophagy signaling pathway, thereby achieving the prevention and treatment effect of DBL on LPS-induced intestinal injury. The experimental results also showed that RES could promote the process of mitophagy, but its effect was weaker than that of DBL at the same dose.

[0074] Example 3 DBL Alleviates LPS-Induced Apoptosis of Leizhou Black Duck Cells

[0075] 1. Experimental Animals and Group Design

[0076] The same method and group design as in Example 1 were used.

[0077] 2. Detection of Apoptosis Rate

[0078] The apoptosis rate in the intestinal tissue of Leizhou black ducks was detected according to the instructions of the apoptosis detection kit (Dojindo Laboratories, Japan). 100 μL of the sample to be tested was taken and 5 μL of AnnexinV, FITC conjugate and 5 μL of PI Solution were added, and incubated in the dark for 15 min. Then 400 μL of 1×AnnexinV Binding Solution was added, and the Annexin V / PI double staining was detected by flow cytometry to determine the apoptosis rate. The results of Annexin V / PI double staining were as Figure 8 shown. Compared with the control group, LPS significantly increased the early and late apoptosis rates of cells and damaged the integrity of the cytoplasmic membrane; compared with the LPS group, the proportion of live cells (LL) in the low- and high-dose DBL intervention groups was significantly increased, and the integrity of the cytoplasmic membrane was increased. Among them, the apoptosis rate of the LPS+DBL (0.04%) group was 6.07%, which was lower than that of the RES group at the same concentration. This indicates that DBL can effectively resist LPS-induced apoptosis of intestinal tissue cells in Leizhou black ducks, and the effect is better than that of RES.

[0079] 3. Quantitative RT-PCR analysis (qRT-PCR) and Western blot analysis (WB)

[0080] Using the same method as in Example 1, the effect of DBL on the apoptosis signaling pathway of intestinal injury Leizhou black duck cells induced by LPS was explored. The proteins detected included BAK1, Caspase-3, Caspase-9, CytC, BAX and BCL-2. The results were as Figure 9 shown. Compared with the control group, the mRNA expressions of BAK1, Caspase-3, Caspase-9 and CytC in the LPS group were significantly increased (P<0.001); compared with the LPS group, DBL could inhibit their expressions. The WB results showed that compared with the control group, the protein expressions of BAK1, Caspase-3, Caspase-9, CytC and the pro-apoptotic protein BAX in the LPS group were significantly increased (P<0.001), and the protein expression of the anti-apoptotic protein BCL-2 was significantly decreased (P<0.001); compared with the LPS group, 0.04% DBL could significantly inhibit the protein expressions of BAK1, Caspase-3, Caspase-9, CytC and the pro-apoptotic protein BAX, and promote the expression of the anti-apoptotic protein BCL-2 (P<0.001). Among them, there was no significant difference in protein expression between the 0.04% DBL treatment group and the control group (P>0.05), and the effect was better than that of RES at the same concentration. This indicates that DBL can inhibit the apoptosis signaling pathway and reduce the apoptosis of intestinal injury Leizhou black duck cells caused by LPS.

[0081] Apoptosis induced by mitochondrial dysfunction is also one of the important factors in LPS-induced intestinal injury. The occurrence of apoptosis is carried out by BAK1 and Bax binding to the outer mitochondrial membrane to form pores. An increase in the expression level of Bax protein can lead to changes in mitochondrial membrane permeability, promoting the release of Cyt-c and Caspase9 from the mitochondrial matrix to the cytoplasm. This process activates the apoptotic signaling pathway, thereby initiating and inducing apoptosis. As pro-apoptotic factors, BAK1 and Bax play important roles in the change of mitochondrial membrane permeability, the production of extranuclear DNA, and the initiation of apoptosis. Bcl-2 has significant anti-apoptotic activity and can bind to BAK1 and Bax to inhibit the apoptotic process. Cyt-c is an essential electron carrier in mitochondria, participating in mitochondrial respiration, and can activate Casepase9, thereby leading to apoptosis. The Caspase family has many members and is a key component in the process of apoptosis. Mitochondrial membrane potential is the electrochemical potential energy necessary for the electron transport chain to synthesize ATP. It will show abnormal changes under the conditions of mitochondrial function obstruction or oxidative stress, thus affecting cell metabolism and mitochondrial quality control. The disruption of mitochondrial membrane potential is the main hallmark of mitochondrial dysfunction and an early event in apoptosis. To explore the effect of DBL on apoptosis of intestinal injury cells induced by LPS in Leizhou black ducks, the present invention detected the change of mitochondrial membrane potential. The experimental results showed that DBL effectively alleviated the decrease of mitochondrial membrane potential. The results of flow cytometry detecting the apoptosis level of intestinal tissue cells showed that 0.04% DBL could significantly reduce the proportion of apoptotic cells and increase the proportion of live cells, and the effect was better than that of RES at the same concentration. The detection and verification of the apoptotic signaling pathway showed that the results of RT-qPCR and Western blot analysis also showed that DBL inhibited the expression of pro-apoptotic related molecules BAK1, Caspase-3, Caspase-9, CytC, and BAX, and promoted the expression of anti-apoptotic molecule BCL-2. In summary, DBL can inhibit mitochondrial membrane potential damage, protect mitochondrial function, thereby reducing the expression of apoptotic proteins, inhibiting apoptosis, and resisting LPS-induced intestinal injury in Leizhou black ducks.

[0082] In summary, the present invention found that DBL can mediate the cGAS / STING and Parkin / PINK1 signaling pathways, improve the activity of antioxidant enzymes, scavenge ROS, restore mitochondrial membrane potential, relieve mitochondrial dysfunction, promote mitophagy and remove damaged mitochondria; it can also mediate the Bax / Bcl-2 signaling pathway, increase the expression of Bcl-2, and decrease the expression of Caspase-3 and Caspase-9, thereby alleviating apoptosis. The present invention expounds the mechanism of action of DBL in protecting cell metabolism, reducing inflammatory response, and relieving LPS-induced intestinal injury in Leizhou black ducks through dual targets, providing a candidate drug for the prevention and treatment of intestinal diseases caused by Gram-negative bacteria in livestock and poultry, contributing to the development of natural drugs that can effectively treat intestinal injury, and contributing to the development of the livestock and poultry breeding industry in China.

[0083] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for relieving intestinal injury in Leizhou black ducks.

2. The application according to claim 1, characterized in that The intestinal injury is intestinal injury caused by lipopolysaccharide.

3. The application according to claim 1, wherein The 3,4-dihydroxybenzylideneacetone effectively maintains the integrity of the intestinal barrier by reducing inflammatory factors and upregulating the expression of intestinal tight junction proteins, thereby relieving the intestinal pathological injury induced by lipopolysaccharide in Leizhou black ducks.

4. Use of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for relieving intestinal mitochondrial dysfunction in Leizhou black ducks.

5. The application according to claim 4, wherein The mitochondrial dysfunction is intestinal mitochondrial dysfunction caused by lipopolysaccharide.

6. The application according to claim 4, characterized in that The 3,4-dihydroxybenzylideneacetone relieves the intestinal mitochondrial dysfunction induced by lipopolysaccharide in Leizhou black ducks by mediating the cGAS / STING and Parkin / PINK1 signaling pathways, increasing the activity of antioxidant enzymes, scavenging ROS, restoring mitochondrial membrane potential, promoting mitophagy and clearing damaged mitochondria.

7. Use of 3,4-dihydroxybenzylideneacetone in the preparation of a drug for inhibiting intestinal cell apoptosis in Leizhou black ducks.

8. The application according to claim 7, wherein The intestinal cell apoptosis is intestinal cell apoptosis caused by lipopolysaccharide.

9. The application according to claim 7, wherein The 3,4-dihydroxybenzylideneacetone inhibits the intestinal cell apoptosis induced by lipopolysaccharide in Leizhou black ducks by regulating the Bax / Bcl-2 signaling pathway, increasing the expression of Bcl-2 and decreasing the expression of Caspase-3 and Caspase-9.

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