Application of tetrandrine in preparation of medicine for preventing and treating pseudorabies of pig

Abstract

The application relates to the technical field of animal infectious disease prevention and control, and particularly discloses application of tetrandrine in preparation of a medicine for preventing and treating pseudorabies of pigs. The application can significantly inhibit the replication efficiency of PRV on Vero cells and IPAM cells. When the concentration of the inhibitor is higher than 4 muM, the progeny virus titer and the virus gene transcription level of PRV are significantly reduced compared with those of a control group, and the pathological changes caused by cell infection are effectively relieved, which indicates that the tetrandrine can effectively inhibit the proliferation of PRV and has a potential application prospect.

Description

Technical Field

[0001] This invention relates to the field of animal infectious disease prevention and control technology, and in particular to the application of tetrandrine in the preparation of drugs for the prevention and treatment of pseudorabies in pigs. Background Technology

[0002] Porcine pseudorabies virus (PRV) belongs to the genus Alphaherpesvirus and is a linear double-stranded DNA virus that causes pseudorabies (PR) in pigs. PRV infection severely impacts the health of pigs and leads to various severe clinical symptoms. In newborn piglets, PRV infection can cause severe encephalitis, accompanied by symptoms such as vomiting and diarrhea, with an extremely high mortality rate. Infected pregnant sows may experience reproductive problems such as abortion. Once pigs are infected with PRV, they can develop persistent infection, carrying the virus for life and making it difficult to eradicate, thus posing a significant threat to the global pig industry, with a particularly prominent impact on my country's pig industry. Furthermore, PRV has a wide host range, infecting various mammals, including canines (such as dogs, foxes, and wolves), felines (such as cats and lions), ruminants (such as cattle and sheep), and rodents (such as rats). In recent years, numerous studies and clinical cases have confirmed that PRV can also infect humans, causing encephalitis and even death, with cases concentrated among those working in the pig industry. This indicates that the prevalence of PRV not only seriously jeopardizes the sustainable development of the aquaculture industry but also poses a significant threat to public health and safety. Currently, the prevention and control of PRV mainly relies on vaccination and strengthened biosecurity measures, and there are no specific treatments available. This situation greatly limits the effective clinical treatment of PRV infection cases. Therefore, how to prevent and treat PRV with drugs is an urgent problem to be solved.

[0003] Tetrandrine is one of the main active ingredients of the Menispermaceae plant *Stephania tetrandra*, belonging to the dibenzylisoquinoline alkaloid class. It possesses various pharmacological activities, including antibacterial, anti-inflammatory, and anticancer effects. Currently, two marketed dosage forms of tetrandrine are available—tablets and injections—and it is commonly used clinically to treat joint pain, lung cancer, and simple silicosis. Furthermore, current research indicates that tetrandrine exhibits activity against African swine fever virus infection in vitro, suggesting its potential application in the antiviral field. Although pharmacological research on tetrandrine is becoming increasingly in-depth, its ability to control porcine pseudorabies virus infection remains unknown. Summary of the Invention

[0004] The present invention aims to provide the application of tetrandrine in the preparation of drugs for the prevention and treatment of pseudorabies in pigs.

[0005] To achieve the above objectives, the technical solution adopted in this invention is as follows: during the infection of IPAM or Vero cells with porcine pseudorabies virus, a compound at a safe concentration for the cells is added and incubated; the compound is tetrandrine.

[0006] The structural formula of the tebufenozide is as follows: The compound tetrandrine was added to the cell culture medium in a specific ratio for use throughout the viral infection process.

[0007] Tetrandrine plays a role in the infection of porcine pseudorabies virus, effectively inhibiting the infection rate and subsequently affecting the viral life cycle, thereby inhibiting viral replication and ultimately effectively treating or preventing porcine pseudorabies virus infection.

[0008] In one preferred embodiment, the tetrandrine or its pharmaceutically acceptable salt is added to a pharmaceutically acceptable carrier or excipient to prepare any pharmaceutically acceptable dosage form; In one preferred embodiment, the dosage form includes powder for injection, capsules, tablets, and suspensions.

[0009] Based on the above scheme, the preferred concentration of tetrandrine is 0.0~15.0 μM.

[0010] Based on the above schemes, further optimization was performed, and the working concentrations of tetrandrine were determined to be 4 μM, 8 μM, and 12 μM.

[0011] Based on the above scheme, the preferred dosage of porcine pseudorabies virus is 5000 TCID50.

[0012] Based on the above schemes, the preferred and selected genotype of porcine pseudorabies virus is a variant strain.

[0013] Based on the above scheme, the preferred and selected cells are African green monkey kidney cells (Vero) and porcine alveolar macrophages (IPAM).

[0014] Based on the above scheme, the preferred cell incubation and culture conditions are 37℃ and cultured in a 5% CO2 incubator for 36 h.

[0015] This invention relates to the application of tetrandrine in the preparation of drugs for the prevention and treatment of porcine pseudorabies. This invention can significantly inhibit the replication efficiency of PRV in Vero cells and IPAM cells. When the inhibitor concentration is higher than 4 μM, compared with the control group, the progeny virus titer and viral gene transcription level of PRV are significantly reduced, and the lesions caused by viral infection are significantly alleviated. This indicates that tetrandrine can effectively inhibit PRV proliferation in cell experiments, providing a new target and approach for the screening of anti-PRV drugs. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0017] Figure 1 This invention relates to the effects of different concentrations of tetrandrine on the activity of IPAM (A) and Vero (B) cells.

[0018] Figure 2 This invention relates to the effect of different concentrations of tetrandrine on PRV-infected cytopathic effects.

[0019] Figure 3 This invention relates to the effect of different concentrations of tetrandrine on PRV progeny viruses.

[0020] Figure 4 This invention relates to the effect of different concentrations of tetrandrine on the transcription levels of the gE and gB genes of PRV-infected cells. Detailed Implementation

[0021] The specific implementation process will be described in detail below with reference to the embodiments of the present invention. It is worth noting that the embodiments described herein are not all embodiments of the present invention. Unless otherwise specified, the test methods and detection techniques described in each embodiment are conventional methods; unless otherwise specified, the reagents and consumables are commercially available.

[0022] 1. Main materials and reagents IPAM cells and Vero cells were both preserved in our laboratory; the PRV mutant strain (HuN-LD-2019) was donated by Professor Wang Aibing of Hunan Agricultural University (isolated according to the description in the literature [1] Lin Y, Tan L, Wang C, He S, Fang L, Wang Z, Zhong Y, Zhang K, Liu D, Yang Q, Wang A. Serological Investigation and Genetic Characteristics of Pseudorabies Virus in Hunan Province of China From 2016 to 2020. Front Vet Sci. 2021, 8: 762326.) and preserved in our laboratory; tetrandrine is a product of Nanjing Baixindenuo Biotechnology Co., Ltd.

[0023] The conventional reagents used in this invention, such as SYBR Green (Novizan) for quantitative fluorescence, cDNA reverse transcription kit (Eric), fetal bovine serum (GBICO), high glucose DMEM medium (GBICO), and 2.5% trypsin (GBICO), are all commercially available.

[0024] Example 1 Tetrandrine cytotoxicity assays against Vero or IPAM cells: Vero or IPAM cells were seeded in 96-well plates. Once the cell density reached 50%–60%, the supernatant was discarded, and different concentrations of tetrandrine and cell maintenance medium were added. The DMSO group served as the control group. Cells were cultured for 36 hours. The OD values ​​of each group were measured using the MTT assay. 490 The relative viability of cells in each group is calculated.

[0025] Example 2 Experiments on the effect of tetrandrine treatment on PRV infection: Seed Vero or IPAM cells into 6-well plates. When the cell density reaches 80%, discard the supernatant and add DMEM medium. Each well is infected with 5000 TCIDs. 50 The PRV mutant strain was adsorbed at 37℃ for 2 hours, the supernatant was discarded, and the strain was washed 3 times with PBS solution. Tetrandrine was diluted to 4 μM, 8 μM, and 12 μM using cell maintenance medium and added to different cell wells. After 36 h of culture, the cytopathic effects of each group were observed. Subsequently, total RNA was extracted from the cells for RT-qPCR to determine the transcriptional levels of PRV-gE and PRV-gB genes in each group; the supernatant was collected for determining the viral titer.

[0026] (1) Cell RNA extraction and RT-qPCR detection The main steps are as follows: 1) After cell infection with the virus, total RNA was extracted using the Trizol method, and 1 μg of RNA was reverse transcribed into cDNA. RT-qPCR primers for the target genes were designed using the online software Primer-Blast (Table 1). All primers were synthesized by Changsha Qingke Biotechnology Co., Ltd. Using the GAPDH gene as an internal control, the relative expression of each detected gene in the cells was verified by qPCR. The qPCR reaction system included 5.0 μL of 2×SYBR Green premix, 0.2 μL each of upstream and downstream primers, 1.0 μL of cDNA template, and 3.6 μL of RNase-free water; the reaction conditions were 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. After the reaction, based on the Ct values ​​corresponding to the target gene and the GAPDH gene in each group, 2... -△△ct The relative expression levels of each gene were analyzed using a statistical method, and the results were statistically analyzed and plotted using GraphPad Prism 9.0 software.

[0027] Table 1 (2) Virus titer determination Vero cells were seeded in 96-well plates. Once the cell density reached 40%–50%, subsequent experiments could be performed. The viral supernatant from each group was serially diluted 10-fold (10-fold, 10-fold, ...). 2 times, 10 3 times......10 8 (Multiple doses), each dilution of virus was inoculated into cells at 100 μL per well, with 8 replicates and a negative control included. The 96-well plates were incubated for 1 h, after which the supernatant was discarded, the cells were washed three times with PBS, and cell maintenance medium was added. After 72 h of incubation, the cells in each well were observed for cytopathic effects, and the number of wells with cytopathic effects in each dilution group was counted. The viral titer was calculated using the Reed-Muench method. See Figure 1 Low concentrations of tetrandrine have little effect on the activity of Vero or IPAM cells, while concentrations of tetrandrine exceeding 20 μM have too great an impact on cell activity. Therefore, the concentration of tetrandrine was selected between 4 μM and 12 μM for subsequent cell experiments. See Figure 2This study investigated the effects of different concentrations of tetrandrine on PRV-infected cell cytopathic effects to preliminarily understand whether tetrandrine has an anti-PRV infection effect. After 24 hours of infection with the PRV mutant strain, cells in the DMSO-treated group showed obvious cytopathic effects and large "cytopathic cavities." In contrast, cells in the high-concentration (12.0 μM) tetrandrine-treated group showed almost no cytopathic effects after viral infection. While cells in the 4.0 μM and 8.0 μM inhibitor-treated groups showed cytopathic effects, these were significantly milder than those in the DMSO-treated group. These results indicate that tetrandrine treatment can effectively alleviate PRV-infected cell cytopathic effects, suggesting that this compound has an anti-PRV infection effect.

[0028] See Figure 3 This study preliminarily explored the therapeutic effect of tetrandrine on PRV infection in vitro, investigating the influence of different concentrations of tetrandrine on PRV infection. Tetrandrine effectively reduced the viral titer of infected PRV progeny in a concentration-dependent manner. Further analysis showed that compared with the DMSO group, tetrandrine concentrations above 4.0 μM reduced viral titer by more than 10 times. Tetrandrine also showed effects on the CCC (correlation) of different cell types. 50 The concentration is approximately 30.0 μM, therefore the therapeutic index of tetrandrine against PRV should be at least higher than 7.5, which indicates low toxicity and high efficacy. See Figure 4 The relative transcription levels of viral gE and gB genes in PRV-infected cells were preliminarily determined after treatment with tetrandrine. Tetrandrine treatment significantly inhibited the relative transcription levels of PRV-gB and gE genes in virus-infected cells, and this inhibition was, to some extent, dose-dependent.

[0029] In summary, this invention, using IPAM (porcine alveolar macrophages) and Vero (African green monkey kidney cells) cells as examples, demonstrated that by pretreating cells with tetrandrine and then inoculating them with PRV, the viral titer, PRV-gE gene transcription level, and cytopathic effects were detected. This showed that tetrandrine can effectively inhibit the proliferation efficiency of PRV in host cells, providing new targets and ideas for the screening of anti-PRV drugs.

[0030] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. The application of tetrandrine in the preparation of a drug for preventing and treating pseudorabies in pigs, characterized in that: Treatment with tetrandrine at safe concentrations for cells can significantly inhibit the replication of porcine pseudorabies virus.

2. The application of tetrandrine as described in claim 1 in the preparation of a drug for preventing and treating pseudorabies in pigs, characterized in that: The structural formula of the tetanine is:

3. The application of tetrandrine as described in claim 1 in the preparation of a drug for preventing and treating pseudorabies in pigs, characterized in that, The aforementioned tetrandrine or its pharmaceutically acceptable salt is added to a pharmaceutically acceptable carrier or excipient to prepare any pharmaceutically acceptable dosage form, including tablets, powder for injection, suspensions, and capsules.

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