Kit for the discovery and application of lyso-phosphatidylcholine (lpc) with anticoccidial effect

Abstract

The application provides a lysophosphatidylcholine (LPC) kit with anti-coccidiosis effect, and discloses a diagnosis marker for Eimeria tenella infected chicken, which is characteristic phospholipid and sphingolipid molecules, and specifically phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM). Meanwhile, the application provides a medicine composition for improving the survival rate of chicks infected with Eimeria tenella, which is characterized by containing effective amounts of 18:2 LPA, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC and d18:1 16:0 SM. The application screens characteristic phospholipid and sphingolipid molecules in the cecum tissue of chicken infected with Eimeria tenella for the first time, and verifies the curative effect of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) on acute infection of chicks with Eimeria tenella, which has great market development prospect.

Description

Technical Field

[0001] This invention relates to a novel use of lysophosphatidylcholine (LPC), specifically to the use of lysophosphatidylcholine (LPC) in the preparation of anticoccidial drugs, and a kit for the discovery and application of lysophosphatidylcholine (LPC) with anticoccidial activity. Background Technology

[0002] Coccidiosis in chickens is a highly serious global parasitic disease caused by several species of Eimeria spp. parasitizing the intestines of chickens. It severely impairs chicken growth and development and is one of the most damaging diseases in intensive poultry farming. The consensus in the industry is that "wherever there are chickens, there is coccidiosis." The harm caused by coccidiosis to the poultry industry is mainly manifested in the following ways: First, severely infected chickens experience decreased or complete loss of appetite, diarrhea or even bloody stools, severe emaciation, and ultimately death; chickens that tolerate the infection suffer from stunted growth due to excessive consumption, thus losing their economic value. Second, mildly infected chickens mainly exhibit reduced feed conversion rates, decreased meat and egg quality, and chronic wasting disease, resulting in huge economic losses. Third, coccidiosis infection disrupts the host's intestinal microenvironment, weakening the chicken's immunity and leading to secondary bacterial or viral diseases, causing economic losses to the poultry industry. It has been reported that the global poultry industry suffers economic losses of more than $2 billion annually due to coccidiosis, with drug expenditures for coccidiosis prevention and treatment exceeding $300 million. my country is a major chicken-producing country, ranking among the world's top in terms of chicken population. However, its overall chicken farming and management standards are lower than those of developed countries, resulting in economic losses due to coccidiosis exceeding the world average and causing more severe economic damage than in other countries. Currently, the main methods for controlling coccidiosis are drug treatment and live vaccine immunization. Due to the large genome, complex life cycle, and numerous antigens of coccidia, vaccine research has not yet achieved a breakthrough. Drug treatment involves adding drugs to the diet or drinking water to control coccidiosis. In this case, the entire developmental stage of coccidia is exposed to drug stress, inevitably leading to drug resistance and causing serious drug resistance problems in chicken farms (CHAPMAN, 1984a). Given these factors, new measures are urgently needed to control coccidiosis.

[0003] Phospholipids and sphingolipids are essential components of both the host (cells and organisms) and parasites, playing roles in energy storage, membrane structure, cell signaling, and transcriptional regulation. Phospholipids and sphingolipids exert multiple functions in cell physiology, and imbalances (deficiency and excess) are closely associated with host organ damage and disease, as well as parasite reproduction. To date, there has been no phospholipid and sphingolipid profiling analysis or application in the process of Eimeria tenella infection. Summary of the Invention

[0004] This invention provides a method for detecting the efficacy of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) against acute infection with Eimeria tenella in chicks using liquid chromatography-electrospray tandem mass spectrometry, comprising the following steps:

[0005] 1) Characteristic phospholipid and sphingolipid molecules in chicken cecal tissue infected with Eimeria tenella were screened using liquid chromatography-electrosprayionization tandem mass spectrometry (LC-MS-MS).

[0006] 2) To verify the efficacy of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) against acute infection with Eimeria tenella in chicks.

[0007] Specifically, the present invention provides a diagnostic marker for Eimeria tenella infection in chickens, wherein the marker is specifically: characteristic phospholipids and sphingolipid molecules.

[0008] Furthermore, the markers are specifically phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM).

[0009] On the other hand, the present invention provides the use of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) in the preparation of a drug for treating acute infection of Eimeria tenella in chicks.

[0010] On the other hand, the present invention provides a drug for treating Eimeria tenella in chicks, characterized in that the drug contains effective amounts of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM).

[0011] Furthermore, in the aforementioned drug, the phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) are selected from 18:2 LPA, 18:0 LPA, 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM, respectively.

[0012] On the other hand, the present invention provides a pharmaceutical composition for improving the survival rate of chicks infected with Eimeria tenella, characterized in that the pharmaceutical composition contains effective amounts of 18:2 LPA, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM.

[0013] On the other hand, the present invention provides a pharmaceutical composition for improving the weight gain rate of chicks infected with Eimeria tenella, characterized in that the pharmaceutical composition contains effective amounts of 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:018:1 PC, 16:018:2 PC, and d18:116:0 SM.

[0014] On the other hand, the present invention provides a pharmaceutical composition for reducing intestinal lesions in chicks infected with Eimeria tenella, characterized in that the pharmaceutical composition contains effective amounts of 18:2 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:018:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM.

[0015] On the other hand, the present invention provides a pharmaceutical composition for reducing the production of oocysts in chicks infected with Eimeria tenella, characterized in that the pharmaceutical composition contains effective amounts of 18:0 LPA, 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, and 16:0 18:2 PC.

[0016] On the other hand, the present invention provides the use of 18:1 lysophosphatidylcholine (18:1 LPC) in the preparation of a drug for controlling coccidiosis, wherein the effective therapeutic concentration of the 18:1 LPC is 20 to 30 μM, preferably 25 μM. Attached Figure Description

[0017] Figure 1 Cecal lysophosphatidylcholine (LPC) analysis

[0018] Figure 2 Analysis of cecal sphingomyelin (SM) Detailed Implementation

[0019] Example 1: Screening of biomarkers in an acute infection model of Eimeria tenella in chicks

[0020] 1. Materials and Methods

[0021] 1.1. Acute infection model of Eimeria tenella in chicks

[0022] Five 14-day-old chicks were infected with fresh, tender Eimeria sporulated oocysts at a dose of 3 × 10⁻⁶. 4 One per animal; a healthy control group was also set up. 120 h after coccidiosis infection, the cecum was harvested, and the contents of the cecum were rinsed with PBS. Store in an 80°C ultra-low temperature freezer.

[0023] 1.2 Phospholipid and Sphingolipid Omics Analysis in Cecal Tissue

[0024] The preserved cecum was ground in liquid nitrogen, weighed, and a certain volume of ddH2O was added to obtain a concentration of 100 mg / mL. Two steel balls were added to a test tube, and the tissue was processed using a tissue lysis apparatus (60 Hz, 60 s). 50 μl of homogenate for each sample was added to 950 μl of methanol containing the internal standard. The mixture was vortexed for 1 minute, allowed to stand for 5 minutes at RT, and then centrifuged at 10,000 rpm for 10 minutes at 4 °C. The supernatant was used for LC-MS-MS analysis.

[0025] Phospholipid and sphingolipid analysis in cecal tissue was performed using a liquid chromatography system equipped with an autosampler and an API QTrap® 4500 mass spectrometer (Applied Biosystems / MDS SCIEX, Forster City, CA, USA) (I-class Acquity ultra-high performance liquid chromatography, Waters, Milford, MA, USA).

[0026] The liquid chromatography parameters were optimized according to reference 1. Lipids were separated using a short C18 HPLC column (5 μm, 2.1 mm ID × 20 mm, TR-0121-C185, Higgins Analytical, Southborough, MA, USA) in negative ion multiple reaction monitoring (MRM) mode. Lipids were separated using a CSH C18 column (1.7 μm, 2.1 mm ID × 100 mm, Waters, Milford, MA, USA) in positive ion MRM mode. Flow components, flow rates, and gradient programs are shown in Tables 1 and 2.

[0027] Table 1. Flow composition, flow rate, and gradient program in multiple reaction monitoring (MRM) mode for negative ions.

[0028]

[0029] Table 2. Flow composition, flow rate, and gradient program in positive ion MRM mode.

[0030]

[0031] The mass spectrometry parameters were set and validated as described above. Curtain gas (CUR) 25, collision gas (CAD) medium, ion source gas 1 (GS1) 45, ion source gas 2 (GS2) 50, electrospray voltage 5500 (positive ion MRM mode) or -4500 (negative ion MRM mode), and temperature 500℃ or 250℃. These parameters were set to obtain the most abundant correlated ions.

[0032] Table 3 lists the mass spectrometry conditions [MRM transition pair, declustering potential (DP), collision energy (CE), internal standard] and validation data [coefficient of determination (R2), limit of detection (LOD), intra-day coefficient of variation (CV) (%) and inter-day coefficient of variation (%)].

[0033] Table 3

[0034]

[0035] The instrument's control, data acquisition, and data processing are all performed by a PC using the software Analyst 3.0 (AppliedBiosystems / MDS SCIEX, Forster City, CA, USA).

[0036] All experiments were independently repeated at least three times. All data were derived from at least three samples per group and are expressed as mean ± SD. Student's t-tests were performed between the simulated and infected groups using GraphPad Prism v9.0 (La Jolla, CA, USA). *P < 0.05, **P < 0.01. Multivariate statistical analyses, including principal component analysis (PCA) and orthogonal least partial square discriminant analysis (OPLS-DA), were performed using SIMCA v14.1 (Umetrics, Umea, Sweden). Furthermore, the importance of the projected variables (VIP) was calculated in the OPLS-DA model. Differentially expressed lipids were screened based on VIP (VIP > 1) and p-values ​​from univariate analyses (P < 0.05).

[0037] The results show (e.g.) Figure 1 and Figure 2As shown in the figure, on day 5 post-infection, significant changes were observed in the levels of lysophosphatidylcholine (18:2 LPC, 20:3 LPC, 20:4 LPC, and 20:5 LPC) and sphingomyelin (d18:1 16:1 SM, d18:1 16:0 SM, d18:1 18:0 SM, d18:1 21:0 SM, d18:2 23:0 SM, d18:1 23:0 SM, d18:1 24:2 SM, and d18:1 24:1 SM) in the cecum of infected chicks. Compared with the control group, the levels of lysophosphatidylcholine (LPC) and sphingomyelin (SM) in the cecum of chicks infected with Eimeria tenella were generally significantly reduced. Given the important role of these lipids in the structure and physiological function of the cecum, this trend in their changes is closely related to cecal damage, hemorrhage, and inflammation caused by Eimeria tenella infection. These suggest that they could serve as biomarkers and intervention targets for Eimeria tenella infection.

[0038] Example 2: Evaluation of the efficacy of different phospholipids and sphingolipids in treating Eimeria tenella infection.

[0039] (1) Dilution of LPA, LPC, and SM:

[0040] The purchased 18:2 lysophosphatidic acid (18:2 LPA), 18:0 LPA, 20:4 LPA, 16:0 lysophosphatidylcholine (16:0 LPC), 18:1 LPC, 22:0 LPC, 16:0 18:1 phosphatidylcholine (16:0 18:1 PC), 16:0 18:2 PC, and d18:1 16:0 sphingomyelin (d18:1 16:0 SM) (18:2 represents the type of acyl group, i.e., different lysophosphatidic acids or lysophosphatidylcholines) were diluted with DMSO to a storage concentration of 20 mM. Store at 80°C. Dilute with PBS to a working concentration of 25 μM before use.

[0041] (2) Animal experiments:

[0042] Eighty-eight 14-day-old chicks of similar weight were randomly divided into 11 groups: nine drug-treated groups, one infection-free group, and one healthy group, with eight chicks in each group. After grouping, except for the healthy group, the other groups were infected with Eimeria tenella sporulated oocysts at a dose of 50,000 oocysts per chick. The drug-treated groups were orally administered the corresponding drug (concentration of 25 μM) at a dose of 1 mL per chick on the day of infection, and then orally every other day for a total of four times.

[0043] Observe for bloody stools on days 4-5 after coccidiosis infection. Count fecal oocysts (OPG) on days 6-8. Weigh the chickens on day 8, cull them, and score their intestinal lesions.

[0044] The anticoccidial index (ACI) is calculated based on survival rate, weight gain, intestinal lesion score, and oocyst production. Efficacy criteria: ACI < 120 indicates no anticoccidial efficacy; ACI = 120–160 indicates low anticoccidial efficacy; ACI = 160–180 indicates moderate anticoccidial efficacy; ACI > 180 indicates highly effective anticoccidial efficacy.

[0045] 2.3. Phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) have significant alleviating effects on acute Eimeria tenella infection in chicks.

[0046] 2.3.1 Survival rate and weight gain

[0047] The results are shown in Table 4. As can be seen from the table, mortality occurred in the infection-free group, as well as in the 18:0 LPA, 20:4 LPA, and 16:0 LPC groups, with survival rates of 66%, 66%, 83%, and 83%, respectively. The survival rate in the remaining groups was 100%. Regarding weight gain, the weight gain in all infection groups was significantly lower than that in the non-infection group. P <0.05); compared with the untreated group, the 18:2 LPA group showed significantly lower weight gain ( P <0.05), no significant change was observed in the 18:0 LPA group ( P >0.05%, and the weight gain in the other treatment groups was significantly increased ( P <0.05).

[0048] Table 4 Survival rate and weight gain

[0049]

[0050] 2.3.2 Blood in stool and intestinal lesions

[0051] The results are shown in Table 5. As can be seen from the table, the amount of bloody stool and the score of intestinal lesions in all drug-treated groups were significantly less than those in the infection-free group. P<0.05). Regarding intestinal lesions, the 18:1 LPC and d18:1 16:0 SM groups had the mildest lesions (+1.0 and +1.2 points, respectively), followed by the 18:2 LPA, 22:0 LPC, 16:0 LPC, 16:0 18:1 PC, and 16:0 18:2 PC groups, with lesion scores ranging from +1.8 to 2.1. The lesion scores of the 18:0 LPA and 20:4 LPA groups were both 2.5.

[0052] Table 5. Bloody stools and intestinal lesions

[0053]

[0054] 2.3.3 Oocyte production

[0055] The results are shown in Table 6. As can be seen from the table, except for the 18:2 LPA and d18:1 16:0 SM groups, the oocyst yield in the other treatment groups was significantly lower than that in the untreated group. P <0.05), among which the follicle yield was relatively low in the 16:0 18:2 PC, 18:1 LPC, 16:0 LPC, 20:4 LPA, and 16:0 18:1 PC groups, with relative follicle yield ranging from 25.2% to 46%.

[0056] Table 6 Oocyte production

[0057]

[0058] 2.3.4 Anticoccidial Index (ACI)

[0059] The results are shown in Table 7. As can be seen from the table, the ACI of all drug-treated groups was significantly higher than that of the untreated group. Among them, the 18:1 LPC group had the best effect, with an ACI greater than 160 but less than 180, achieving a moderate anticoccidial effect. The ACI of the 18:2 LPA and 18:0 LPA groups was less than 120, showing no anticoccidial effect. The ACI of the other drug-treated groups was between 120 and 150, indicating a low-efficacy anticoccidial effect.

[0060] Table 7. Anticoccidial Index (ACI)

[0061]

[0062] The results show that, in terms of survival rate, the efficacy of 18:2 LPA, 18:1 LPC, 22:0 LPC, 16:018:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM was significantly higher than that of the untreated group after infection. In terms of relative weight gain, the efficacy of 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM was significantly higher than that of the untreated group after infection. In terms of intestinal lesions, the efficacy of 18:2 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM was significantly higher than that of the untreated group after infection. From the perspective of oocyst production, the therapeutic effects of 18:0 LPA, 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, and 16:0 18:2 PC were higher than those in the untreated group. Considering all factors, 25 μM of 18:1 lysophosphatidylcholine (18:1 LPC) showed the best efficacy and was effective within the concentration range of 20 to 30 μM.

[0063] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for improving the survival rate of chicks infected with Eimeria tenella, characterized in that... The pharmaceutical composition contains effective amounts of 18:2 LPA, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:116:0 SM.

2. A pharmaceutical composition for improving the weight gain rate of chicks infected with Eimeria tenella, characterized in that... The pharmaceutical composition contains effective amounts of 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM.

3. A pharmaceutical composition for reducing intestinal lesions in chicks infected with Eimeria tenella, characterized in that... The pharmaceutical composition contains effective amounts of 18:2 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, 16:0 18:2 PC, and d18:1 16:0 SM.

4. A pharmaceutical composition for reducing oocyst production in chicks infected with Eimeria tenella, characterized in that... The pharmaceutical composition contains effective amounts of 18:0 LPA, 20:4 LPA, 16:0 LPC, 18:1 LPC, 22:0 LPC, 16:0 18:1 PC, and 16:0 18:2 PC. 5.18:1 lysophosphatidylcholine in the preparation of drugs for controlling coccidiosis, wherein the effective therapeutic concentration of the 18:1 LPC is 20 to 30 μM.

6. The application as described in claim 5, wherein the effective therapeutic concentration of 18:1 LPC is 25 μM.

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