Use of muscone in preparation of virus inhibitor

Abstract

The present application relates to the field of biological medicine, in particular to a kind of muscone in the application of preparation virus inhibitor.The virus inhibitor of the present application is used for the replication of porcine epidemic diarrhea virus.The present application finds that muscone has no cytotoxicity, and does not directly inactivate virus, but inhibits porcine epidemic diarrhea virus invasion cell by reducing caveolin level.The antiviral effect is remarkable, the mechanism of action is clear, the selectivity is high, and the safety is better.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of muscone in the preparation of viral inhibitors. Background Technology

[0002] Japanese encephalitis and porcine epidemic diarrhea virus are important pathogens that threaten the pig industry. Their high infectivity and high mortality rate in pig herds make them key areas for prevention and control.

[0003] Japanese encephalitis, also known as epidemic encephalitis B (hereinafter referred to as JE), is a zoonotic disease caused by the Japanese encephalitis virus (JEV). According to experimental statistics, approximately 67,900 new cases are reported globally each year, with about 50% occurring in China. JEV belongs to the Flaviviridae family and the Flavivirus genus, and is a neurotropic virus. Its primary target cells are neurons; both direct infection with JEV and the mediated immune inflammatory response can cause neuronal cell death. The viral replication titer in the periphery is crucial in determining whether JEV can cross the blood-brain barrier and enter the central nervous system; therefore, controlling viral replication is essential for intervening in the progression of JE. Currently, the pathogenesis of JE is not fully understood, and there are no effective antiviral drugs for its treatment; clinical treatment mainly focuses on symptomatic and supportive care. Japanese encephalitis is a major disease that seriously harms the pig farming industry. It mainly affects the reproductive performance of sows, such as causing abortion, stillbirth, and mummified fetuses in pregnant sows, and orchitis in boars. It can also cause persistent high fever in adult pigs and fattening pigs, leading to encephalitis in newborn piglets.

[0004] The pathogen of porcine epidemic diarrhea (PEDD) is porcine epidemic diarrhea virus (PEDV), a coronavirus. It was first discovered in the UK in the late 1970s and then spread to other European countries, experiencing occasional outbreaks in the latter half of the 20th century. Since 2010, PEDD has been widespread in southern China, affecting pigs of all ages. Notably, PEDV has spread rapidly throughout the country, with a near 100% mortality rate in newborn piglets. Following its initial report in the US in 2013, a highly pathogenic PEDV emerged and rapidly infected pig herds. Subsequently, the virus spread rapidly in the pig farming industries of Europe, North America, and Asia, posing a serious economic threat to the global pig industry. Based on over 600 tissue and fecal samples collected in nine provinces in East, South, and Central China from 2010 to 2011, the average PEDV infection rate was 58.32%, with PEDV positivity rates exceeding 50% in all provinces. A study of 732 diarrheal samples collected from 16 provinces in my country between 2020 and 2021 showed a PEDV nucleic acid positivity rate of 66.80%. Currently, PEDV has become one of the key pathogens restricting the steady development of my country's pig farming industry, second only to African swine fever virus and porcine reproductive and respiratory syndrome virus in terms of its impact.

[0005] The aforementioned virus is characterized by rapid spread, high mortality, and significant harm to the pig farming industry. Furthermore, existing drug treatments are ineffective. Therefore, finding new and highly effective drugs presents a cost-related technical challenge. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides an application of muscone in the preparation of virus inhibitors, which can serve as a reference for current swine disease prevention and control programs.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the use of muscone in the preparation of a virus inhibitor for inhibiting the replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus.

[0008] In some embodiments, the viral inhibitor inhibits the replication of Japanese encephalitis virus by modulating at least one of the NLRP3 / Caspase-1 signaling pathway, the Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway, and the JNK / ERK / P38 signaling pathway.

[0009] In some embodiments, the viral inhibitor is capable of downregulating the protein levels of at least one of NLRP3, Caspase-1, p-JNL, CleavedCaspase-9, CleavedCaspase-3, and Bax; and / or The viral inhibitor can upregulate the protein level of Bcl-2.

[0010] In some embodiments, the viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits the Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway; or The viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits the NLRP3 / Caspase-1 signaling pathway; or The viral inhibitor is a Japanese encephalitis virus inhibitor that inhibits the JNK / ERK / P38 signaling pathway.

[0011] In some embodiments, the viral inhibitor inhibits the replication of porcine epidemic diarrhea virus by downregulating the level of Caveolin-1 protein; and / or The viral inhibitor suppresses the replication of porcine epidemic diarrhea virus by inhibiting viral invasion of cells.

[0012] In some embodiments, the virus inhibitor is a porcine epidemic diarrhea virus inhibitor that inhibits Caveolin-1 protein expression.

[0013] In some embodiments, the virus inhibitor is a drug for the prevention, diagnosis, and treatment of Japanese encephalitis and / or swine epidemic diarrhea.

[0014] In some implementations, the virus inhibitor is used to prepare at least one of pharmaceuticals, animal foods and their additives, feed, cosmetics, and perfumes.

[0015] Secondly, the present invention also provides a virus inhibitor comprising: a first active ingredient comprising: muscone, wherein the virus inhibitor is used to inhibit the replication of Japanese encephalitis virus and / or porcine epidemic diarrhea virus.

[0016] In some embodiments, the virus inhibitor further includes: one of an excipient, a carrier, and a second active ingredient, wherein the second active ingredient can enhance the activity of the first active ingredient or reduce the adverse reactions of the first active ingredient; and / or The viral inhibitors are used to prevent, diagnose, and treat Japanese encephalitis and / or swine epidemic diarrhea.

[0017] Experiments have verified that muscone does not exhibit cytotoxicity against BHK-21, HMC3, SK-N-SH, and Vero-81 cells, and it cannot directly inactivate Japanese encephalitis virus (JEV) or porcine epidemic diarrhea virus (PEDV). Muscone exerts its antiviral effect by inhibiting viral protein expression, thereby suppressing viral replication. Furthermore, muscone exerts its antiviral effect against JEV by inhibiting signaling pathways such as NLRP3 / Caspase-1, Caspase-9 / Caspase-3 / Bax / BCL-2, and JNK / ERK / P38, and by reducing caveolin levels to inhibit PEDV invasion of cells.

[0018] Furthermore, muscone not only possesses a novel antiviral mechanism, but also exhibits remarkable antiviral activity against JEV at a concentration of 10 μM, demonstrating extremely strong antiviral effects. At a concentration of 20 μM, it also shows significant antiviral activity against PEDV, exhibiting extremely strong anti-porcine epidemic diarrhea virus infection activity.

[0019] In addition, muscone has low residue, no pollution, is easily absorbed by animal bodies, has a high biological metabolic rate, and is excreted without pollution when used as an antiviral drug.

[0020] Therefore, muscone has significant antiviral effects against Japanese encephalitis virus and porcine epidemic diarrhea virus, with a clear mechanism of action, high selectivity, and good safety. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 The results are for the cytotoxicity assay of muscone, where A is BHK-21, B is SK-N-SH, C is HMC3, and D is Vero-81.

[0023] Figure 2 The results show the virus inactivation activity of muscone against JEV, with ns representing no significant difference.

[0024] Figure 3 The results show the inhibitory effect of muscone on JEV replication. A represents BHK-21+JEV, B represents SK-N-SH+JEV, and C represents HMC3+JEV.

[0025] Figure 4 The results of this study investigate the inhibitory mechanism of muscone on JEV from the NLRP3 / Caspase-1 signaling pathway.

[0026] Figure 5The results of this study investigate the inhibitory mechanism of muscone on JEV from the JNK / ERK / P38 signaling pathway.

[0027] Figure 6 The results of this study investigate the inhibitory mechanism of muscone on JEV from the Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway.

[0028] Figure 7 The results show the viral inactivation activity of muscone against PEDV; ns represents no significant difference.

[0029] Figure 8 The results show the inhibitory effect of muscone on PEDV replication.

[0030] Figure 9 The results show the effect of muscone on the degradation of caveolin.

[0031] Figure 10 The test results show that muscone inhibits EGFP-PEDV replication.

[0032] Figure 11 The experimental procedure for Example 4 is as follows.

[0033] Figure 12 The results are the clinical index statistics for Example 4.

[0034] Figure 13 The survival rate statistics are for Example 4.

[0035] Figure 14 The results of neuroinflammatory marker detection are from Example 4.

[0036] Figure 15 The expression of interferon-stimulated genes IFIT1, Mx1, and Mx2 in Example 4 is shown.

[0037] Figure 16 This shows the expression of the JEV-C gene in Example 4.

[0038] Figure 17 The results show the viral titer of JEV in the brain, liver, spleen, and heart tissues of mice in Example 4. Detailed Implementation

[0039] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] <Muscone> Muscone is an active ingredient obtained by distillation extraction of musk, the dried secretion from the musk sac of mature male musk deer (Moschus berezovskii Flerov or Moschus moschiferus L.). It is the main aroma component of musk. The chemical name of muscone is 3-methylpentadecanone, and its molecular formula is C1. 16 H 30 O, with a molecular weight of 238.42, is a pale yellow, oily liquid with a distinctive odor. It is very slightly soluble in water but miscible with ethanol. Its molecular structure is as follows: .

[0041] Japanese encephalitis virus Japanese encephalitis virus (JEV) belongs to the genus Flaviviridae in the family Flaviviridae. It is a neurotropic virus that primarily targets neurons. Direct infection with JEV and the immune inflammatory response mediated by it can both cause neuronal cell death, leading to Japanese encephalitis (JE).

[0042] JEV is an enveloped virus with a particle diameter of approximately 35-40 nm. Its genome is a single-stranded positive-sense RNA, about 11 kb in length. The genome structure includes a 5' untranslated region (5'UTR), approximately 95 nucleotides, containing a type I cap structure (m 7 G 5 'ppp 5 The 'A' region is involved in the regulation of translation initiation; the open reading frame (ORF) encodes a polyprotein (approximately 10,299 nucleotides), which, after cleavage by host and viral proteases, forms 10 mature proteins; the 3' untranslated region (3'UTR): approximately 574-585 nucleotides, lacking a poly(A) tail, can form conserved secondary structures (such as stem-loop structures), and is associated with viral replication and virulence. The genomic RNA encodes a polyprotein on the endoplasmic reticulum (ER) membrane, which is cleaved into three structural proteins (Capsid, prM, and Envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Based on the E gene sequence, JEV is classified into five genotypes (GI-GV). In China, GI and GIII types are predominantly prevalent, with GI type exhibiting higher replication efficiency and pathogenicity than GIII type, possibly related to the amino acid differences at positions 55 (D / E) and 65 (E / D) of the NS2B protein, which affect protease activity. JEV has only one serotype, and there is cross-immune protection among different genotypes.

[0043] <Porcine Epidemic Diarrhea Virus> Porcine Epidemic Diarrhea Virus (PEDV) is a major pathogen causing epidemic diarrhea in pigs. Belonging to the genus Coronavirus in the family Coronaviridae, it poses a significant threat to piglets, especially newborns, and is one of the most important viral infectious disease pathogens of concern in the global pig industry. PEDV is a single-stranded positive-sense RNA virus. The viral particles are spherical or elliptical, enveloped, and covered with rod-shaped spikes (spike protein, S protein). This structure is closely related to the virus's host specificity, pathogenicity, and immunogenicity. The S protein is a key antigen that induces the production of neutralizing antibodies and is also a major target for vaccine development. PEDV primarily infects pigs through the oral-gastrointestinal route. After entering the body orally, the virus specifically recognizes and binds to receptors (such as aminopeptidase N) on the surface of small intestinal epithelial cells (especially the villous epithelial cells of the duodenum and jejunum), subsequently invading the cells and replicating within them. Viral replication leads to degeneration, necrosis, and shedding of the epithelial cells of the small intestinal villi, causing the villi to become shorter, thicker, or even fused. This severely impairs the absorption function of the intestines, leading to impaired absorption of water and electrolytes, resulting in severe diarrhea and dehydration. This is the core mechanism of PEDV pathogenesis.

[0044] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0045] The experimental materials used in the following examples include, but are not limited to: Porcine Epidemic Diarrhea Virus (PEDV), Japanese Encephalitis Virus (JEV), BHK-21 (hamster kidney fibroblasts), HMC3 (human microglia), and Vero-81 (African green monkey kidney cells) were all preserved by our laboratory research group.

[0046] The CCK-8 kit was purchased from Wuhan Sewell Biotechnology Co., Ltd.

[0047] Muskone (CAS: 541-91-3) was purchased from Selleck.

[0048] Example 1: Cytotoxicity test of muscone BHK-21, HMC3, SK-N-SH, and Vero-81 cells were seeded into 96-well plates at a density of approximately 80%. After three washes with PBS, cells were cultured in thymol at concentrations of 2 µM, 10 µM, 20 µM, 50 µM, and 100 µM. Cells treated with DMSO served as controls. After 24 h, cells were washed, and OD values ​​were measured at 450 nm using a microplate reader according to the manufacturer's instructions. Cytotoxicity was expressed by the formula: Cell viability (%) = (OD experimental wells - OD blank wells) / (OD control wells - OD blank wells) × 100%. Each experiment was performed in triplicate, and data were presented as mean ± standard deviation (SD).

[0049] like Figure 1 As shown, the experimental groups treated with different concentrations of muscone showed no significant difference in cell activity within a certain drug concentration range (e.g., less than or equal to 20 μM), indicating that muscone at concentrations less than or equal to 20 μM does not have significant cytotoxicity to BHK-21, HMC3, SK-N-SH, Vero-81 cells.

[0050] Example 2: Study of muscone against JEV 1. Test of the virus-inactivating activity of muscone against JEV JEV virus solution was mixed with different concentrations of muscone, with a total volume of 1 mL, and incubated at 37°C for 2 h in increments of concentration. The mixture was then inoculated into cells, followed by incubation at 37°C for another 2 h. Unbound virus was removed by washing with PBS. Control cells were infected with the same amount of JEV at a muscone concentration of 0. After incubation at 37°C for 24 h, RNA samples were collected and reverse-engineered into cDNA. JEV genome copy number was detected by q-PCR. For statistical analysis, the fold change in JEV mRNA level in the control group (0 muscone concentration) was taken as 1, and JEV genome copy number data for other experimental groups were calculated.

[0051] The results are as follows Figure 2 As shown, muscone did not have a direct inactivation effect on JEV at concentrations of 0-20 μM, confirming that muscone inhibits JEV activity not through direct virus inactivation.

[0052] 2. Test on the inhibitory effect of muscone on JEV replication BHK-21, SK-N-SH, and HMC3 cells were respectively thrombized at 1×10⁻⁶ 5Cells were seeded at a density of 100 cells / well into 12-well plates. After reaching approximately 80% cell density, the cells were washed twice with serum-free DMEM, seeded with JEVs, and incubated at 37°C for 2 hours. Cell maintenance medium of different concentrations of thymol was then added. Cell samples were collected after 24 hours and subjected to Western blotting. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0053] The results are as follows Figure 3 As shown, compared with the experimental group with a muscone concentration of 0 μM, the expression level of JEV NS5 in cells treated with different concentrations of muscone was significantly reduced, proving that muscone can significantly inhibit the replication of JEV virus.

[0054] 3. Study on the inhibitory mechanism of muscone on JEV (1) NLRP3 / Caspase-1 signaling pathway With 1×10 5 HMC3 cells were seeded into 12-well plates at a density of approximately 80%. After washing twice with serum-free DMEM, JEVs were inoculated and cultured at 37°C for 2 hours. Cell maintenance medium containing 0-20 μM thymol was then added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0055] The results are as follows Figure 4 As shown, JEV infection significantly activates the NLRP3 / Caspase-1 pathway 24 hours after infection. Compared with the untreated group, the NLRP3 / Caspase-1 protein level decreased in the 20 μM muscone treatment group, indicating that muscone significantly inhibits the activation of the NLRP3 / Caspase-1 pathway.

[0056] (2) JNK / ERK / P38 signal path With 1×10 5 HMC3 cells were seeded into 12-well plates at a density of approximately 80%. After washing twice with serum-free DMEM, JEVs were inoculated and cultured at 37°C for 2 hours. Cell maintenance medium containing 0-20 μM thymol was then added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0057] The results are as follows Figure 5As shown, JEV infection significantly activates the JNK / ERK / P38 pathway 24 hours later. Compared with the untreated group, the p-JNL protein level decreased in the 20 μM muscone treatment group, indicating that muscone significantly inhibits the activation of the JNK / ERK / P38 pathway.

[0058] (3) Caspase-9 / Caspase-3 / Bax / Bcl-2 signaling pathway With 2×10 5 SK-N-SH cells were seeded into 12-well plates at a density of 10 cells / well. After reaching approximately 80% cell density, the cells were washed twice with serum-free DMEM, inoculated with JEVs, and cultured at 37°C for 2 hours. Cell maintenance medium containing 0-20 μM thymol was then added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was not seeded with JEVs, and thymol was replaced with DMSO.

[0059] The results are as follows Figure 6 As shown, the Caspase-9 / Caspase-3 / Bax / Bcl-2 pathway was significantly activated 24 hours after JEV infection. Compared with the untreated group, the 20 μM muscone treatment group showed decreased levels of leached Caspase-9, leached Caspase-3, and Bax proteins, and increased levels of Bcl-2 protein, indicating that muscone significantly inhibited the activation of the Caspase-9 / Caspase-3 / Bax / Bcl-2 pathway.

[0060] Example 3: Study on muscone against PEDV 1. Test of the viral inactivation activity of muscone against PEDV PEDV virus solution was mixed with different concentrations of muscone, with a total volume of 1 mL, and incubated at 37°C for 2 h in increments of concentration. The mixture was then inoculated into cells, followed by incubation at 37°C for another 2 h. Unbound virus was removed by washing with PBS. Control cells were infected with an equal amount of JEV at a muscone concentration of 0. Cells were incubated at 37°C until more than 80% CPE was formed, and the supernatant was collected after freeze-thaw cycles for testing. Virus inactivation was subsequently assessed using TCID50.

[0061] The results are as follows Figure 7 As shown, muscone did not directly inactivate PEDV at concentrations of 0-20 μM, confirming that muscone inhibits PEDV activity not through direct viral inactivation.

[0062] 2. Test on the inhibitory effect of muscone on PEDV replication Vero-81 cells were loaded at a rate of 1×10⁻⁶. 5Cells were seeded at a density of 100 cells / well into 12-well plates. After reaching a density of approximately 80%, the cells were washed twice with serum-free DMEM, seeded with PEDV, and incubated at 37°C for 2 hours. The liquid in the wells was discarded, and cell maintenance medium of different concentrations of muscone was added. Cell samples were collected after 24 hours and subjected to Western blotting. The control group (represented as "con" in the figure) had a muscone concentration of 0 μM.

[0063] The results are as follows Figure 8 As shown, compared with the control group with a 0 μM musketone concentration, the expression level of PEDV-N in cells treated with different concentrations of musketone was significantly reduced, demonstrating that musketone can significantly inhibit the replication of PEDV virus.

[0064] 3. Study on the inhibitory mechanism of muscone on PEDV (1) Muscone can degrade caveolin With 1×10 5 Vero-81 cells were seeded into 12-well plates at a density of approximately 80%. After washing twice with serum-free DMEM, cells were inoculated with PEDV and cultured at 37°C for 2 hours. The liquid in the wells was discarded, and cell maintenance medium containing 0-20 μM thymol was added. Cell samples were collected after 24 hours for Western blot analysis. The control group (represented as "con" in the figure) was either inoculated with PEDV (represented as "+") or not inoculated with PEDV (represented as "-"), and thymol was replaced with DMSO.

[0065] Caveolin-1 is a type of structural protein on the membrane of host cells (such as small intestinal epithelial cells of pigs). Its main functions include participating in cell signal transduction, endocytosis (such as cholesterol transport), and maintaining the structure of the cell membrane.

[0066] The results are as follows Figure 9 As shown, caveolin-1 protein was significantly degraded under the action of muscone. Compared with the untreated group, the caveolin-1 protein level decreased in the 20 μM muscone-treated group, indicating that muscone can degrade caveolin-1 at the protein level, thereby reducing the expression level of caveolin-1.

[0067] (2) Muscone inhibits PEDV invasion of cells by degrading caveolin. Cell crawling slides were placed in 12-well cell culture plates, and cell suspensions were inoculated. Once the Vero-81 cell density reached 60%, the cells were washed with PBS and pretreated with 2µM, 10µM, and 20µM muscarin at 37°C for 1 h. The supernatant was discarded, and the cells were washed with PBS and incubated with EGFP-overexpressing PEDV (EGFP-PEDV, MOI 0.05) for 2 h. EGFP is a green fluorescent protein. The cells were washed again and treated with different concentrations of muscarin for 24 h. The supernatant was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, followed by washing with PBS. The cells were blocked with 1% BSA at room temperature for 30 min. Caveolin-1 monoclonal rabbit anti-antibody was incubated overnight at 4°C. The antibody was then discarded, and the cells were washed with PBS. Cy3-labeled goat anti-rabbit IgG was incubated at room temperature in the dark for 1 h, followed by washing with PBS. Nuclear staining was performed with DAPI, followed by incubation at room temperature in the dark for 10 min, and then washing with PBS. Add 1 mL of PBS to each well, observe and capture images using an OLYMPUS laser scanning confocal microscope, and process the images. A negative control group and a blank control group were set up. Compared to the experimental groups, the negative control group (CON) did not receive muscardine treatment after EGFP-PEDV inoculation, while the blank control group (Mock) did not receive EGFP-PEDV inoculation after muscardine pretreatment, but was subsequently treated with muscardine.

[0068] The results are as follows Figure 10 As shown, fluorescence observation reveals that EGFP fluorescence weakens with increasing muscone concentration, indicating that muscone inhibits EGFP-PEDV replication in a dose-dependent manner. Figure 9 The results presented showed that muscone significantly degraded Caveolin-1 expression. Since the main functions of Caveolin-1 are to participate in cell signal transduction, endocytosis (such as cholesterol transport), and cell membrane structure maintenance, it is speculated that muscone inhibits PEDV invasion of cells by degrading caveolin, thereby inhibiting EGFP-PEDV replication.

[0069] Example 4 Animal Experiment 1. Protective effect of muscone on JEV-infected mice (1) Forty C57 mice aged 4-6 weeks were randomly divided into four groups: a blank control group, a single-drug control group, a JEV group, and a JEV+drug group, with 10 mice in each group. The blank control group was represented as DMSO+DMEM (also represented as DMSO), indicating that DMSO was administered and DMEM was administered at the time of viral infection. The single-drug control group was represented as Muscone+DMEM (also represented as Muscone), indicating that Muscone was administered and DMEM was administered at the time of viral infection. The JEV group was represented as DMSO+JEV (also represented as JEV), indicating that DMSO was administered and JEV was administered at the time of viral infection. The JEV+drug group was represented as Muscone+JEV, indicating that Muscone was administered and JEV was administered at the time of viral infection. Specifically, the Muscone dosage was 12 mg / kg per day, the DMEM dosage was 100 μL per day, and the JEV viral titer was 2 × 10⁻⁶. 6 The concentration of PFU / mL and the amount of virus administered is 100 μL.

[0070] For detailed experimental procedures, please refer to... Figure 11 As shown, mice were exposed to the virus on day 1, and administered the drug via intraperitoneal injection on days 3, 4, and 5, at a dose of 12 mg / kg per day. The mice were observed for disease progression, and their behavior, weight changes, and mortality were recorded daily after exposure.

[0071] On day 6 after virus inoculation, five mice were randomly selected from each group. Their brain tissue and important organs were taken, a portion of which was homogenized, RNA and protein samples were extracted, and the homogenate supernatant was collected. The expression of pro-inflammatory factors and interferon was detected by real-time RT-PCR, and the viral titer was detected by plaque detection. The remaining five mice were continuously observed until day 21 after virus inoculation, and their weight changes and survival status were recorded to draw survival curves.

[0072] Summarize the daily recorded weight changes, condition, and survival status, and calculate the clinical indices and survival rates for each group.

[0073] Figure 12 As shown in the figure, the clinical index statistics indicate that 60% of the mice in the JEV group developed clinical symptoms on day 5 after infection; the proportion of mice in the Muscone+JEV group with symptoms was lower than that in the DMSO+JEV group, and some even showed no clinical symptoms. On day 8, all mice in the Muscone+JEV group developed clinical symptoms, but only 20% did. On day 9, all mice in the JEV group died, and 20% of the mice in the Muscone+JEV group developed symptoms compared to day 8. By day 15, 50% of the mice in the Muscone+JEV group showed symptoms, while the remaining 50% remained asymptomatic until day 21. This demonstrates that muscone can alleviate clinical symptoms in mice infected with JEV.

[0074] Figure 13 The survival rate statistics are shown in the figure. Mice in the JEV group began dying on day 6, while the blank control group (…) Figure 13 The Chinese text refers to DMSO and the control group treated alone (DMSO). Figure 13 Mice treated with muscone (represented as 'Muscone') survived until the end of the experiment. In the muscone+JEV group, 20% of the mice died on day 8, with a mortality rate of 50% by the end of the experiment. All mice in the JEV group died on day 9. These results indicate that muscone treatment can improve the survival rate of mice infected with JEV.

[0075] (2) Muscone can inhibit neuroinflammation induced by JEV infection in mice.

[0076] Five groups of mouse brain tissue were randomly selected, homogenized, and RNA was extracted. The expression of pro-inflammatory cytokines TNF-α, IL-1β, and CCL-5 was detected by quantitative real-time RT-PCR. like Figure 14 As shown, in mouse brain tissue, the expression levels of TNF-α, IL-1β, and CCL-5 genes in experimental group 1 were significantly lower than those in the JEV group, indicating that muscone can inhibit neuroinflammation induced by JEV infection in mice.

[0077] (3) Muscone can inhibit JEV replication in mice by upregulating the transcription level of interferon-stimulated genes.

[0078] Five groups of mouse brain tissue were randomly selected, homogenized, and RNA was extracted. The expression of interferon-stimulated genes IFIT1, Mx1, and Mx2 was detected by quantitative real-time RT-PCR.

[0079] like Figure 15 As shown, in mouse brain tissue, the expression levels of TIFIT1, Mx1, and Mx2 in the Muscone+JEV group were significantly higher than those in the JEV group; Figure 16 As shown, the expression level of the JEV-C gene in experimental group 2 was significantly lower than that in the JEV group, indicating that muscone can inhibit the replication of JEV in mice by upregulating the transcription level of interferon-stimulated genes.

[0080] (4) The viral load in the brain and peripheral blood of mice in the muscone group was significantly reduced. Brain, liver, spleen, and heart tissues were aseptically collected from mice. 0.3g of tissue was added to 1mL of PBS and homogenized in a cryogenic homogenizer. The supernatant was collected. The supernatant was filtered through a 0.22μm filter under aseptic conditions. The filtered homogenate was then subjected to TCID45. 50 The test is performed to determine the amount of virus present. Simply put, it involves diluting the product by 10... -1 Up to 10 -8A supernatant sample was added to BHK-21 cells at a concentration of 1:1. 96-well plates were incubated for 4 to 5 days. Each dilution was added to 4 to 8 wells. Cytopathic effect (CPE) was observed under an optical microscope, and TCID was calculated using Reed-Muench and Karber methods. 50 .

[0081] like Figure 17 As shown, the viral titers of JEV in the brain, liver, spleen, and heart tissues of mice in the Muscone+JEV group were significantly lower than those in the JEV group, indicating that muscone can reduce the viral load of JEV in the brain and periphery of mice after JEV infection.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of muscone in the preparation of a virus inhibitor for inhibiting the replication of porcine epidemic diarrhea virus.

2. The use according to claim 1, characterized in that, The virus inhibitor is a porcine epidemic diarrhea virus inhibitor that inhibits Caveolin-1 protein expression.

3. The use according to any one of claims 1-2, characterized in that, The virus inhibitor is a drug used for the prevention and treatment of swine epidemic diarrhea.

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