New application of new drug RIG-1 agonist-cnidium lactone 6-site derivative for treating PRRS (porcine reproductive and respiratory syndrome)

Abstract

The invention discloses a novel application of an osthole 6-site derivative. The cnidium lactone 6-site derivative disclosed by the invention can be used for effectively inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) and activating a porcine retinoic acid-induced gene I-like receptor (RIG-I). The cnidium lactone 6-site derivative disclosed by the invention is rich in source, simple in preparation process, low in production cost and extremely remarkable in anti-PRRSV activity (P is less than 0.0001), and is expected to be developed into a new drug for treating porcine reproductive and respiratory syndrome (PRRS).

Description

Technical Field

[0001] This application is supported by Tianjin Youth Science and Technology Talents (Third Level) Project, Project Number: QN20230327.

[0002] The invention belongs to the field of preventive veterinary medicine, and in particular relates to a 6-position derivative of osthole. The invention also relates to the application of the compound in treating porcine reproductive and respiratory syndrome and activating RIG-1. Background Art

[0003] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV can infect pigs of all stages and show symptoms mainly related to reproductive disorders and respiratory diseases. PRRSV infection can cause miscarriage, stillbirth or weak fetuses in pregnant sows, fever, loss of appetite and respiratory diseases in growing and nursery pigs, and can cause death in pigs in severe cases. The prevention and control of PRRSV currently mainly relies on vaccines, but due to the high variability and easy recombination of PRRSV itself, the commercial vaccines currently on the market cannot provide good cross-protection for the prevention and control of PRRSV, which also increases the difficulty of clinical prevention and control. Therefore, it is of great significance to develop an efficient, green and healthy compound to directly or indirectly resist PRRSV.

[0004] Osthole 6-derivatives are a class of small molecule compounds that can be isolated from Zanthoxylum spinulosum. Literature reports that the compound has anti-insect activity [1] . The inventors studied the chemical composition of the methanol extract of the root bark of Zanthoxylum spinulosum and isolated a 6-position derivative of osthole as shown in Formula Ⅰ. After screening for anti-PRRSV activity, it was found for the first time that the compound has a significant inhibitory effect on PRRSV and can activate the porcine retinoic acid-induced gene I-like receptor (RIG-1) and upregulate its immune level. This indicates that the 6-position derivative of osthole may be further developed into a new drug RIG-1 agonist for the treatment of porcine reproductive and respiratory syndrome. There are no literature reports on the use of the 6-position derivative of osthole for the treatment of porcine reproductive and respiratory syndrome. Summary of the invention

[0005] To achieve the above purpose, the technical contents disclosed in the present invention are as follows: A 6-position derivative of osthole, having a structure shown in Formula I:

[0006] The present invention further discloses the use of osthole derivatives at position 6 in the preparation of drugs for treating porcine reproductive and respiratory syndrome virus (PRRSV). The experimental results show that after 31.25µM osthole derivatives at position 6 were used to treat PRRSV-infected cells for 4 hours, the viral load showed a very significant decrease (P < 0.0001), indicating that osthole derivatives at position 6 can significantly inhibit the proliferation of PRRSV.

[0007] The present invention further discloses the use of the 6-position derivative of osthole in the preparation of drugs for activating porcine retinoic acid-induced gene I-like receptor (RIG-I) and improving immunity; experimental results show that the expression of RIG-1 protein in the group treated with 31.25µM 6-position derivative of osthole significantly increased.

[0008] The present invention mainly investigates a 6-position derivative of osthole and its new use in resisting PRRSV and activating RIG-1. The key point is to find that the 6-position derivative of osthole has a very significant inhibitory effect on PRRSV and a new use for activating RIG-1. The difficulty of the invention lies in the rich raw material source of the 6-position derivative of osthole and the discovery of significant anti-PRRSV and RIG-1 activation effects.

[0009] The positive effects of the 6-position derivative of osthole disclosed in the present invention and its effects on anti-PRRSV and activation of RIG-1 compared with the prior art are: 1. The 6-position derivative of osthole of the present invention has abundant sources, simple preparation process and low production cost.

[0010] 2. The 6-position derivative of osthole of the present invention has extremely significant resistance to PRRSV and can activate RIG-1. It can be used in the treatment of porcine reproductive and respiratory syndrome and is expected to be developed into a new drug for the treatment of porcine reproductive and respiratory syndrome.

[0011] 3. The 6-position derivative of osthole of the present invention has the characteristics of high efficiency, low cost, green and safe, and is expected to be developed into a new green anti-PRRSV drug RIG-1 agonist.

[0012] References [1] W. Zhang, W. Guan, Z. Geng, Y. Wang, X. Pang, C. You, S. Du, Two new coumarins from Zanthoxylum dimorphophyllum spinifolium and their feedingdeterrent activities against Tribolium castaneum, Industrial Crops and Products 143 (2020) 111889. https: / / doi.org / 10.1016 / j.indcrop.2019.111889. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Anti-PRRSV effects of osthole 6-derivatives (Formula I) under different modes of action; Figure 2 : The anti-PRRSV effect of osthole 6-derivative (Formula I) at different action times; Figure 3 : The anti-PRRSV effect of osthole 6-derivative (Formula I) at different concentrations; Figure 4 : Effects of osthole 6-derivatives (Formula I) on RIG-1 expression. DETAILED DESCRIPTION

[0014] The present invention is described below through specific implementation schemes. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the implementation scheme should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are only limited by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation schemes, without departing from the essence and scope of the present invention, also fall within the scope of protection of the present invention. The raw materials used in the present invention are abundant and can be collected, and the reagents used are commercially available. References to the extraction method of the 6-position derivative of osthole [1] . Example

[0015] Anti-PRRSV effects of osthole 6-derivatives in different modes of action Experimental methods: The in vitro antiviral effects of traditional Chinese medicine can be divided into inhibition of virus proliferation, blocking virus infection and direct virus killing according to different administration methods. Inhibition of virus proliferation refers to the use of drugs to treat cells after the virus infects the cells for a certain period of time; blocking of virus infection refers to pre-treating cells with drugs before infecting cells with viruses; direct virus killing refers to infecting cells after the virus and drugs act for a certain period of time.

[0016] The PAM cells were revived in a 12-well plate and cultured in an incubator for 24 hours until they were in good condition. 1MOI of TJ-C6 strain was added for infection for 1 hour, and the virus solution was recovered and rinsed with PBS 2-3 times, and then the cells were treated accordingly according to different administration methods. After incubation for 24 hours, the cells were repeatedly frozen and thawed at -80℃ for 3 times, the cell supernatant RNA was extracted, and the PRRSV viral load was detected using a real-time fluorescence quantitative kit.

[0017] Experimental results: According to the three modes of action of traditional Chinese medicine in vitro antiviral research, cells were treated differently to screen out the best mode of action of osthole derivatives at the 6th position to exert antiviral effects. The results of the determination of viral load showed that the osthole derivatives at the 6th position had a good anti-PRRSV effect in the inhibitory mode (P < 0.0001) ( Figure 1 ), the subsequent experiments will be carried out using this treatment method, see Figure 1 . Example

[0018] The best action time of osthole 6-derivatives against PRRSV Experimental method: PAM cells were revived in 12-well plates and cultured in an incubator for 24 hours until they were in good condition. 1MOI of TJ-C6 strain was added for infection for 1 hour, and the virus solution was recovered and rinsed with PBS 2-3 times, followed by adding 2% cell maintenance solution containing the maximum safe concentration of osthole 6 derivatives, and incubated for 4, 6, 8, 12, and 24 hours, respectively, and repeatedly frozen and thawed at -80℃ for 3 times, and the cell supernatant RNA was extracted, and the PRRSV viral load was detected using a real-time fluorescence quantitative kit.

[0019] Experimental results: According to the best mode of action screened out, we continued to explore the best action time of the 6-position derivative of osthole, set the time nodes to 4, 6, 8, 12, and 24 hours, and detected the viral load at each action time point. The results showed ( Figure 2 ), except for the viral load at 12h, which was not significantly different from the viral control group, the viral load at other time points decreased, and the viral load was the lowest when the action time was 4h (P < 0.0001). Therefore, 4h was taken as the optimal action time of the 6-position derivative of osthole (see Figure 2 ). Example

[0020] The Optimal Concentration of Osthole 6-Derivatives against PRRSV Experimental method: PAM cells were revived in 96-well plates and cultured in an incubator for 24 hours until they were in good condition. 1MOI of TJ-C6 strain was added for infection for 1 hour, and the virus solution was recovered and rinsed with PBS 2-3 times, followed by adding 2% cell maintenance solution containing different concentrations of Chinese medicine monomer compounds, incubated for 24 hours, and repeatedly frozen and thawed at -80℃ for 3 times, and the cell supernatant RNA was extracted, and the PRRSV viral load was detected using a real-time fluorescence quantitative kit.

[0021] Experimental results: Four drug concentrations were set at the maximum safe concentration, namely 62.5µM, 31.25µM, 15.6µM and 7.8µM, and the virus-infected cells were treated with compound 3 for 4 hours in a way that inhibited viral proliferation, and the viral load was detected. Except for the 7.8µM treatment group, which had no significant decrease in viral load, the viral loads of the other three concentrations of treatment groups decreased to varying degrees, and the viral load under the 31.25µM treatment was extremely significantly reduced (P < 0.0001). Figure 3 . Example

[0022] Osthole 6-position derivatives activate RIG-1 Experimental method: Four groups were set up, including control group, PRRSV virus group, osthole 6-derivative group, and PRRSV+coumarin compound group. After culturing cells in each group for 24 hours, the supernatant was discarded. The control group was added with DMEM equal to the virus solution of C6 strain with MOI=1, adsorbed for 1 hour, then the DMEM was discarded, and 2% cell maintenance medium was added to culture for 4 hours; the PRRSV virus group was added with C6 strain virus solution with MOI=1, adsorbed for 1 hour, then the virus solution was discarded, and 2% cell maintenance medium was added to culture for 4 hours; The 6-position derivative of osthole group was added with DMEM equal to the C6 strain virus liquid with MOI=1, adsorbed for 1 hour, then discarded the DMEM, added with 2% cell maintenance medium containing 31.25μM coumarin compound 3 and cultured for 4 hours; the PRRSV+coumarin compound 3 group was added with C6 strain virus liquid with MOI=1 for 1 hour, then discarded the virus liquid, added with 2% cell maintenance medium containing 31.25μM osthole 6-position derivative and cultured for 4 hours. Protein samples were extracted from each group for Western Blot experiment. The results are shown in Figure 4 .

[0023] Experimental results: Comparing the RIG-1 expression levels in the four groups, with β-actin as the internal reference, the expression of RIG-1 was significantly increased after the treatment with 31.25 μM osthole derivative at the 6th position, indicating that the osthole derivative at the 6th position can activate the expression of RIG-1 and play an immunomodulatory role.

[0024] After the preferred embodiments are described in detail, those familiar with the technology can clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above patent application, and any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention. The present invention is not limited to the example implementation methods given in the specification.

Claims

1. Use of a 6-position derivative of osthole in the preparation of a drug for treating porcine reproductive and respiratory syndrome virus (PRRSV).

2. Use of a 6-position derivative of osthole in the preparation of a drug for activating porcine retinoic acid-induced gene I-like receptor (RIG-I) to improve pig immunity.

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