Use of a HIF-1α inhibitor in the preparation of a medicine against PEDV infection

Abstract

The application discloses application of a HIF-1 alpha inhibitor in preparation of a medicine for resisting PEDV infection, and belongs to the technical field of biotechnology.It is proved through experiments that the HIF-1 alpha protein inhibitor can significantly inhibit the activation of the HIF-1 alpha signal path, can significantly inhibit the replication of PEDV, reduces cell damage, and plays an antiviral role.The application lays a theoretical foundation for developing a new type of anti-PEDV medicine, and provides a new direction for prevention and treatment of porcine epidemic diarrhea.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of an HIF-1α inhibitor in the preparation of a drug for treating PEDV infection. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious gastrointestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). It is characterized by diarrhea, vomiting, and high mortality in suckling piglets and is one of the major pathogens seriously threatening the healthy development of the pig industry. PEDV belongs to the genus *Coronavirus-α*, with a genome length of approximately 28 kb. PEDV can infect pigs of different ages, but suckling piglets show the most severe symptoms and a mortality rate as high as 100%.

[0003] Although commercially available vaccines against PEDV are widely used, the immature immune systems of suckling piglets cannot provide effective immune protection, and even immunized piglets are susceptible to infection. The small intestine of piglets is the primary target organ for PEDV, and intestinal epithelial cells (IECs) are the target cells for PEDV infection. IECs exist in a unique "physiological hypoxic" environment because they are distributed between the microvascular-rich, oxygen-rich submucosa and the severely hypoxic intestinal lumen, resulting in a steep oxygen gradient within the IEC cell layer. Therefore, IECs have developed a unique hypoxic response mechanism to adapt to this "physiological hypoxic" environment. Hypoxia-inducible factor-1α (HIF-1α), as the most important transcriptional regulator in the hypoxic response mechanism, is continuously expressed in IECs and plays a crucial role in maintaining intestinal metabolic homeostasis.

[0004] Therefore, based on the specific hypoxic environment of the target organs infected by PEDV, exploring an effective new anti-PEDV drug is of great significance for the prevention and treatment of porcine epidemic diarrhea. Summary of the Invention

[0005] The purpose of this invention is to provide an application of an HIF-1α inhibitor in the preparation of drugs against PEDV infection, thereby addressing the problems existing in the prior art. This invention experimentally demonstrates that the use of an HIF-1α protein inhibitor can significantly inhibit the activation of the HIF-1α signaling pathway and significantly inhibit PEDV replication, reducing cell damage and exerting an antiviral effect. This invention lays the theoretical foundation for the development of novel anti-PEDV drugs and provides a new direction for the prevention and treatment of porcine epidemic diarrhea.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of an HIF-1α inhibitor in the preparation of a drug for treating PEDV infection.

[0008] Furthermore, the anti-PEDV infection includes resistance to PEDV infection of cells.

[0009] Furthermore, the cells include African green monkey kidney epithelial cells.

[0010] Furthermore, the HIF-1α inhibitor includes Bay 87-2243.

[0011] Furthermore, the HIF-1α inhibitor inhibits PEDV replication.

[0012] Furthermore, the HIF-1α inhibitor inhibits PEDV transcription and replication by preventing PEDV from entering the host cell nucleus.

[0013] Furthermore, the drug is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients, with the HIF-1α inhibitor as the active ingredient.

[0014] Furthermore, the drug is an oral medication.

[0015] Furthermore, the dosage forms of the drug include granules, solutions, pills, ointments, and tablets.

[0016] The present invention discloses the following technical effects:

[0017] This invention, through in vitro and in vivo experiments, found that PEDV infection promotes the expression of hypoxia-inducible factor-1α (HIF-1α), indicating that HIF-1α protein is a potential antiviral target. Further verification revealed that the use of HIF-1α protein inhibitors significantly inhibited the activation of the HIF-1α signaling pathway and significantly suppressed PEDV replication, reducing cell damage and exerting an antiviral effect.

[0018] This invention lays the theoretical foundation for the development of novel anti-PEDV drugs and provides a new direction for the prevention and treatment of porcine epidemic diarrhea. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The expression level (A) and transcription level (B) of HIF-1α in Vero E6 cells at different time points after infection with PEDV;

[0021] Figure 2 The expression level (A) and transcription level (B) of HIF-1α in the small intestine tissue of newborn piglets 48 h after PEDV infection;

[0022] Figure 3 The effect of PEDV infection on HIF-1α nuclear translocation is shown in the following figures: A represents the Western blot results of HIF-1α in the cytoplasm and nucleus of Vero E6 cells after PEDV infection; B represents the IFA results of HIF-1α in the nucleus of Vero E6 cells after PEDV infection; and C represents the IFA results of HIF-1α in the nucleus of small intestinal tissue cells of newborn piglets after PEDV infection.

[0023] Figure 4 The inhibitory effect of Bay 87-2243 on PEDV infection is shown in the following figures: A represents the effect of Bay 87-2243 on HIF-1α expression after PEDV infection, as detected by Western blot; B represents the results of quantitative real-time PCR detection of HIF-1α after 24 hours of PEDV infection and Bay 87-2243 addition; C represents the results of plaque assay after 24 hours of PEDV infection and Bay 87-2243 addition; and D represents the results of CCK8 assay of cell viability after treating Vero E6 cells with different concentrations of Bay 87-2243 for 24 hours. Detailed Implementation

[0024] Various exemplary embodiments 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, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention, through in vitro and in vivo experiments, found that PEDV infection promotes the expression of hypoxia-inducible factor-1α (HIF-1α), indicating that HIF-1α protein is a potential antiviral target. Further verification revealed that the use of HIF-1α protein inhibitors significantly inhibited the activation of the HIF-1α signaling pathway and significantly suppressed PEDV replication, reducing cell damage and exerting an antiviral effect.

[0030] In a specific embodiment of the present invention, the HIF-1α inhibitor used is Bay 87-2243, CAS No.: 1227158-85-1, molecular formula: C 26 H 26 F3N7O2, the structural formula is as follows:

[0031]

[0032] The newborn piglets used in this invention were purchased from a pig farm in Binzhou City, Shandong Province. Molecular and immunological diagnostic methods confirmed that they were negative for porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), porcine transmissible gastroenteritis virus (TGEV), and porcine PDCoV. The cells used in the in vitro experiments were the VeroE6 cell line of African green monkey kidney epithelial cells, purchased from ATCC and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Shandong Academy of Agricultural Sciences. The test strain was isolated from and preserved by the inventors from the intestines of diarrheal piglets at a pig farm in Zibo City, Shandong Province. It was identified as the GIIb subtype and designated as the PEDV SDZB strain.

[0033] Example

[0034] I. Experimental Methods

[0035] 1. Effects of PEDV infection on the HIF-1α signaling pathway in host cells

[0036] After Vero E6 cells were infected with PEDV (0.1 MOI), cell samples were collected at specified times (6h, 12h, 24h, and 48h). The dynamic changes in HIF-1α transcription level, protein level, and nuclear translocation were observed by quantitative real-time PCR, Western blot, and indirect immunofluorescence staining (IFA).

[0037] Newborn piglets in the challenge group were orally administered 2 mL EDV SDZB strain (1×10⁻⁶) 6 TCID 50 PEDV virus solution ( / mL) was administered orally to newborn piglets in the negative control group, along with the same dose of DMEM culture medium. The piglets' health status was observed until obvious diarrhea appeared (approximately 48-72 hours), at which point they were euthanized, and small intestinal tissue was collected. Total RNA and total protein were extracted from the tissue, and the effects of PEDV infection on HIF-1α transcription and protein levels in the small intestinal tissue were detected by quantitative real-time PCR and Western blot.

[0038] 2. Effects of Bay 87-2243 on the HIF-1α signaling pathway and PEDV infection

[0039] After PEDV infection of Vero E6 cells, the HIF-1α signaling pathway inhibitor Bay 87-2243 (10 μM) was added. Cells and supernatant were collected 24 h after infection. The effects of Bay 87-2243 on the HIF-1α signaling pathway and PEDV infection were observed by quantitative real-time PCR, Western blot, and plaque assay.

[0040] 3. Indirect immunofluorescence staining test

[0041] After dewaxing, piglet small intestine tissue sections underwent antigen retrieval and peroxidase elimination. The sections were perforated and perforated with 0.4% Triton X-100 for 15 min, followed by blocking with 5% bovine serum albumin (BSA) for 2 h. After blocking, mouse PEDV-N protein monoclonal antibody (1:100) and rabbit HIF-1α protein polyclonal antibody (1:100) were added to the sections, and incubated overnight at 4°C. After washing with primary antibody, goat anti-mouse secondary antibody labeled with CoraLite488 and goat anti-rabbit secondary antibody labeled with CoraLite594 were added, and the sections were incubated at 37°C for 1 h. Then, DAPI (0.5 μg / mL) was added to stain the cell nuclei for 5 min. After washing, anti-fluorescence attenuation mounting medium was added, and confocal microscopy was performed for observation and analysis.

[0042] 4. Quantitative Real-Time PCR

[0043] Extraction and reverse transcription of total RNA from tissue / cell samples to obtain cDNA: Tissue samples were homogenized in RNA-easy Isolation Reagent to release total RNA. RNA was then extracted using the Trizol method (cell samples were extracted directly without homogenization). Reverse transcription was performed using Novizan's HiScript II Reverse Transcriptase kit to obtain cDNA from the tissue samples.

[0044] Quantitative real-time PCR was performed according to the instructions for the Novizan ChamQ SYBR qPCR Master Mix reagent. Primer sequences are shown in Table 1. The target and internal control genes were each tested in triplicate, using 2... -ΔΔCT Analyze the experimental data.

[0045] Table 1 Primer sequences

[0046]

[0047]

[0048] 5. Western blot

[0049] Tissue samples were homogenized in RIPA lysis buffer (containing 1% PMSF) to release proteins (cell samples were directly lysed in RIPA lysis buffer). After centrifugation, the supernatant was collected, and protein concentration was determined according to the BCA protein concentration assay kit instructions. Sample proteins were diluted and adjusted according to their concentration, then denatured by SDS and subjected to SDS-PAGE gel electrophoresis. Subsequently, the proteins were transferred to methanol-activated PVDF membranes using a rapid wet transfer apparatus. After blocking with 5% skim milk powder for 2 hours, the PVDF membranes containing the target bands were incubated overnight at 4°C with mouse PEDV-N protein monoclonal antibody (1:1000), rabbit HIF-1α protein polyclonal antibody (1:1000), and HRP-labeled GAPDH antibody (1:5000), respectively. After washing five times with TBST, HRP-labeled goat anti-mouse secondary antibody (1:8000) and HRP-labeled goat anti-rabbit secondary antibody (1:8000) were added, and the membranes were incubated at room temperature for 2 hours. After washing, the protein bands were exposed and observed using an automated chemiluminescence imaging system with ECL chemiluminescence solution. The resulting protein bands were analyzed using ImageJ software.

[0050] II. Experimental Results

[0051] 1. PEDV infection promotes HIF-1α expression in host cells.

[0052] PEDV infection promotes the activation of the HIF-1α signaling pathway in cells: Quantitative real-time PCR, Western blot, and indirect immunofluorescence staining assays revealed that 24 hours after PEDV infection, the protein and transcriptional levels of HIF-1α in Vero E6 cells significantly increased. Figure 1 A and Figure 1 (B).

[0053] PEDV infection promotes HIF-1α expression in the small intestine of newborn piglets: Quantitative real-time PCR and Western blot analysis revealed that PEDV infection of newborn piglets via the digestive tract increased HIF-1α expression in the small intestine. Figure 2 A and Figure 2 (B).

[0054] PEDV infection promotes HIF-1α nuclear translocation: Vero E6 cells were infected with PEDV (0.1 MOI), cell samples were collected, nuclear and cytoplasmic proteins were separated, and the dynamic changes in HIF-1α nuclear translocation were detected. Results showed that 12 h after PEDV infection, HIF-1α levels in the cytoplasm significantly decreased, while HIF-1α levels in the nucleus significantly increased. Figure 3(A); The effect of PEDV infection on HIF-1α nuclear translocation was observed by IFA (blue, DAPI; green, PEDVN protein; red, HIF-1α). The results showed that 24 h after PEDV infection, more HIF-1α accumulated in the cell nucleus. Figure 3 (B). Meanwhile, IFA was used to observe the nuclear translocation of HIF-1α in newborn piglets. PEDV infection promoted the accumulation of HIF-1α in the nucleus of small intestinal epithelial cells of newborn piglets. Figure 3 The results (C) indicate that PEDV infection promotes the activation of the HIF-1α signaling pathway in the small intestine.

[0055] 2. Effects of HIF-1α inhibitors on PEDV infection

[0056] After PEDV infection, 10 μM Bay 87-2243 was added. Cells were collected 24 hours later, and Western blot analysis revealed that Bay 87-2243 inhibited HIF-1α expression and significantly suppressed PEDV N protein expression. Figure 4 (A); Through quantitative real-time PCR and plaque assays, it was found that Bay 87-2243 significantly inhibited the transcription of PEDVN protein and the release of progeny viruses during tooth eruption. Figure 4 (B and C); Bay 87-2243 was used to treat Vero E6 cells at different concentrations for 24 h, and the effect on cell viability was detected by CCK8 assay. It was found that there was no cytotoxicity at 10 μM. Figure 4 (D). The above results indicate that Bay87-2243 can inhibit the infection of porcine epidemic diarrhea virus.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of an HIF-1α inhibitor in the preparation of a drug for treating PEDV infection, characterized in that, The HIF-1α inhibitor is Bay 87-2243.

2. The application according to claim 1, characterized in that, The anti-PEDV infection includes resistance to PEDV infection of cells.

3. The application according to claim 2, characterized in that, The cells include African green monkey kidney epithelial cells.

4. The application according to claim 1, characterized in that, The HIF-1α inhibitor inhibits the transcription and replication of PEDV.

5. The application according to claim 1, characterized in that, The HIF-1α inhibitor inhibits PEDV replication by preventing PEDV from entering the host cell nucleus.

6. The application according to claim 1, characterized in that, The drug is prepared by adding pharmaceutically acceptable excipients to the HIF-1α inhibitor as the active ingredient.

7. The application according to claim 6, characterized in that, The drug is an oral medication.

8. The application according to claim 7, characterized in that, The dosage forms of the drug include granules, solutions, pills, ointments, and tablets.

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