Use of methyl bardoxolone in the preparation of a medicament for inhibiting respiratory syncytial virus infection
By using methylbadoxolone to inhibit the IKKβ/NF-κB pathway, drugs in various dosage forms were prepared, solving the problem of the lack of effective treatments for RSV infection and inflammatory damage in existing technologies, and achieving the effect of effectively inhibiting RSV infection and reducing lung damage.
Patent Information
- Application Number
- CN202310407222.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-14
- Publication Date
- 2025-11-18
- Estimated Expiration
- 2043-04-14
AI Technical Summary
There is a lack of effective drugs for treating respiratory syncytial virus (RSV) infection, especially for the inflammatory damage caused by RSV, and existing drugs such as ribavirin and pallizumab have toxic side effects and limitations.
Bardoxolone Methyl is used as a novel small molecule compound to inhibit respiratory syncytial virus infection and inflammatory factor expression by inhibiting the IKKβ/NF-κB pathway. It is prepared into dosage forms such as tablets, capsules, powders, granules, injections or oral liquids.
It effectively inhibits RSV infection and the expression of related inflammatory factors, reduces lung damage, and provides a new drug strategy for the treatment of RSV infection. Furthermore, the therapeutic effect of methylbardoxolone is comparable to that of ribavirin, with fewer side effects.
Smart Images
Figure CN116473979B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of methylbardoxolone in the preparation of drugs for inhibiting respiratory syncytial virus infection. Background Technology
[0002] Respiratory syncytial virus (RSV) is one of the most common viral pathogens affecting the respiratory tract, spreading widely worldwide. RSV is a leading cause of bronchiolitis and pneumonia in premature infants and young children, affecting approximately 60% of preschool children globally. It is estimated that 94,600–149,400 children under the age of five die annually from RSV infection worldwide. RSV is also a significant pathogen causing respiratory infections in the elderly (over 65 years of age) and immunocompromised individuals. According to the World Health Organization, approximately 33 million people worldwide are infected annually, with over 3.4 million experiencing severe infections requiring hospitalization. Of these hospitalized patients, 10–31% require intensive care, and 3–17% require mechanical ventilation. Currently, there is a lack of mature vaccines and effective treatments for RSV infection. Therefore, in-depth research into drugs and mechanisms for treating RSV infection is particularly important. Previous clinical specimens and in vitro experiments revealed that RSV infection mediates innate immune damage to Toll-like receptor 3 (TLR3) and Toll-like receptor 7 (TLR7), and stimulates the NF-κB signaling pathway to produce a large number of inflammatory mediators, such as IL-6, IL-8, IL-10, IL-13, IL-32, TNF-α, etc., which increases inflammatory factors and aggravates lung damage.
[0003] Although RSV has been known for over 60 years, treatment options remain very limited. To date, only two drugs are approved by the U.S. Food and Drug Administration (FDA) for the prevention and treatment of RSV: inhaled ribavirin and palilizumab. Ribavirin is a nucleoside analog and a broad-spectrum antiviral drug. Early studies showed it could rapidly clear RSV infection, alleviate symptoms, and shorten hospital stays. However, other opinions suggest that ribavirin does not reduce mortality or the duration of mechanical ventilation in severely ill patients after RSV infection. Furthermore, ribavirin can cause hemolysis, birth defects, and cardiovascular toxicity, leading to increasing skepticism regarding its use. Palilizumab is a humanized mouse monoclonal antibody targeting the respiratory syncytial virus fusion protein (RSV F). It is only used to prevent severe lower respiratory tract infections caused by RSV in children. It significantly reduces hospitalizations, length of hospital stays, and mortality, but due to its rapid metabolism, it requires re-injection every six months, is expensive, and is not available in my country.
[0004] Currently, there is a lack of mature vaccines and effective treatments for RSV infection. Therefore, there is an urgent need to develop an effective treatment for RSV infection. Summary of the Invention
[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the application of methylbardosolone in the preparation of drugs to inhibit respiratory syncytial virus (RSV) infection. This invention observes the pathological effects of RSV infection in BEAS-2B cells using an inverted microscope, detects the cytotoxicity and antiviral effects of methylbardosolone using the CCK8 assay, detects the expression of RSV, IL-6, IL-8, IKKβ, and NF-κB mRNA using reverse transcription polymerase chain reaction (RT-PCR), and detects the expression of RSV F, p-IKKβ, p-NF-κB, and IL-6 proteins using Western blot. The results show that the molecular mechanism of methylbardosolone's anti-RSV infection is related to the IKKβ / NF-κB pathway, confirming that methylbardosolone inhibits RSV infection and the expression of RSV inflammatory factors by inhibiting the IKKβ / NF-κB pathway.
[0006] A first aspect of the present invention provides the use of methylbardoxolone in the preparation of a medicament for inhibiting respiratory syncytial virus infection.
[0007] Specifically, the use of methylbardoxolone in the preparation of drugs that inhibit respiratory syncytial virus infection.
[0008] Bardoxolone Methyl (BXM) is a novel small molecule compound, a semi-synthetic triterpenoid oleanolic acid derivative, with CAS number 218600-53-4. It possesses antioxidant, anti-inflammatory, anti-proliferative, and anticancer effects. Bardoxolone directly targets Keap1 and IKKβ. Its binding to Keap1 disrupts cysteine residues, leading to the release of Nrf2, which induces antioxidant and anti-inflammatory responses. Furthermore, bardoxolone can directly bind to Cys-179 in the IKKβ activation ring, inhibiting NF-κB activation and thus suppressing downstream pro-inflammatory pathways.
[0009] The use of methylbardoxolone in the preparation of drugs that inhibit respiratory syncytial virus infection by inhibiting the IKKβ / NF-κB pathway.
[0010] Application of methylbardoxolone in the preparation of drugs that inhibit the expression of respiratory syncytial virus inflammatory factors by inhibiting the IKKβ / NF-κB pathway.
[0011] The application of methylbardoxolone in the preparation of drugs that inhibit respiratory syncytial virus infection and the expression of respiratory syncytial virus inflammatory factors by inhibiting the IKKβ / NF-κB pathway.
[0012] Preferably, the concentration of methylbardoxolone is 0.01-20 mg / kg / d.
[0013] More preferably, the concentration of methylbardoxolone is 0.1-10 mg / kg / d.
[0014] More preferably, the concentration of methylbardoxolone is 1-5 mg / kg / d.
[0015] More preferably, the concentration of the methylbardoxolone is 1-3 mg / kg / d.
[0016] More preferably, the concentration of methylbardoxolone is 2.5-3 mg / kg / d.
[0017] Preferably, the dosage form of the drug is at least one of tablets, capsules, powders, granules, injections, and oral liquids.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] (1) This invention proposes that methylbardoxolone can be used to prepare drugs that inhibit respiratory syncytial virus infection. Currently, there is no relevant research on the molecular mechanism of methylbardoxolone in the treatment of RSV infection and inflammatory damage. This invention establishes RSV-infected cell and mouse models, observes the pathological effect of BEAS-2B cells infected with RSV using an inverted microscope, detects the cytotoxicity and antiviral effect of methylbardoxolone using CCK8 reagent (Cell Counting Kit-8), detects the expression of RSV, IL-6, IL-8, IKKβ and NF-κB mRNA by reverse transcription polymerase chain reaction (RT-PCR), and detects the expression of RSV F, p-IKKβ, p-NF-κB and IL-6 proteins by Western blot. This invention is the first to confirm the pharmacodynamics of methylbardoxolone in alleviating lung inflammatory damage caused by RSV infection through the IKKβ / NF-κB pathway. Methylbardoxolone can be used to prepare drugs that inhibit respiratory syncytial virus infection, in order to provide promising drugs, therapeutic targets and strategies for the clinical diagnosis and treatment of RSV.
[0020] (2) This invention utilizes methylbardosolone to prepare a drug that inhibits respiratory syncytial virus infection and the expression of respiratory syncytial virus inflammatory factors by inhibiting the IKKβ / NF-κB pathway. The therapeutic effect can be regulated by controlling the concentration of methylbardosolone. The therapeutic effect of methylbardosolone at a dose of 1 mg / Kg / d is comparable to that of ribavirin at a dose of 40 mg / Kg / d. The therapeutic effect of methylbardosolone at a dose of 3 mg / Kg / d is the best in RSV-infected mice. Attached Figure Description
[0021] Figure 1 This is a schematic diagram of the experimental method used in in vitro experiments;
[0022] Figure 2 This is a schematic diagram of the grouping and technical route for in vivo experiments;
[0023] Figure 3 Image showing RSV infection in Hep-2 cells observed by scanning electron microscopy;
[0024] Figure 4 Images showing RSV infection of Hep-2 and BEAS-2B cells under a microscope;
[0025] Figure 5 This diagram illustrates the inhibitory effect of methylbardoxazoline on RSV in BEAS-2B cells by inhibiting the IKKβ / NF-κB pathway.
[0026] Figure 6 The effect of TPCA-1 on RSV infection in BEAS-2B cells is shown in the figure.
[0027] Figure 7 A graph showing the changes in mouse body weight in different groups;
[0028] Figure 8 Images of hematoxylin-eosin (HE) staining of mouse lung tissue from different groups;
[0029] Figure 9 Pathological scoring images of mouse lung tissue from different groups;
[0030] Figure 10 Figure 1 shows the results of virus titer determination in mouse lung tissue from different groups.
[0031] Figure 11 This figure illustrates how methylbardoxolone inhibits RSV infection and the production of related inflammatory factors in mice by suppressing the IKKβ / NF-κB pathway. Detailed Implementation
[0032] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.
[0033] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.
[0034] I. Experimental Methods
[0035] 1. Cell experiments:
[0036] To investigate whether methylbardoxone can inhibit RSV infection and whether methylbardoxone can inhibit RSV-induced inflammatory damage through the IKKβ / NF-κB pathway, an RSV-infected cell model was established to study the effect and molecular mechanism of methylbardoxone against RSV infection through the IKKβ / NF-κB pathway.
[0037] (1) BEAS-2B (human bronchial epithelial cells) and Hep-2 (human laryngeal cancer epithelial cells, RSV amplification vector) cells were purchased from American Type Culture Collection (ATCC), and the cell lines were in the 2nd-3rd generation.
[0038] (2) Preparation of a cell RSV infection model:
[0039] BEAS-2B and Hep-2 cells were resuscitated and passaged. RSV amplification was performed in Hep-2 cells, and the amplified TCID was measured. 50 Then with 500 TCID 50 RSV infection BEAS-2B.
[0040] (3) To explore the molecular mechanisms related to the IKKβ / NF-κB pathway:
[0041] ① Using BEAS-2B cells as the experimental subject, according to... Figure 1 Divided into: normal control group, 500 TCID 50 RSV infection group, 500 TCID 50 RSV+0.1μM methylbardoxazoline group, 500TCID 50Four groups were included: RSV + 0.3 μM methylbardoxazoline. The pathological effects of RSV infection on BEAS-2B cells were observed using an inverted microscope. The cytotoxicity and antiviral effects of methylbardoxazoline were detected using the CCK8 assay. The mRNA expression of RSV, IL-6, IL-8, IKKβ, and NF-κB was detected by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of RSV F, p-IKKβ, p-NF-κB, and IL-6 was detected by Western blot.
[0042] ② A control experiment was conducted using the IKKβ-specific inhibitor TPCA-1 and methylbardoxolone. The experiment was divided into: a normal control group and a 500 TCID group. 50 RSV infection group, 500 TCID 50 RSV + 0.3 μM methylbardoxazoline group, 500 TCID 50 The RSV+0.3μMTPCA-1 group was used to detect RSV mRNA expression by RT-PCR, which proved that not all IKKβ inhibitors can inhibit RSV infection through the IKKβ / NF-κB pathway.
[0043] 2. Animal experiments
[0044] (1) Preparation of mouse RSV infection model:
[0045] ① Healthy, female SPF-grade BALB / c mice (6-8 weeks old, weighing 20-25g) were bred according to... Figure 2 Mice were randomly divided into six groups: wild-type group (i.e., untreated mice, serving as blank control), RSV infection group, BXM (3 mg / Kg / d) group, RSV infection + low-dose BXM (1 mg / Kg / d) group, RSV infection + high-dose BXM (3 mg / Kg / d) group, and RSV infection + ribavirin (40 mg / Kg / d) positive control group (ribavirin RIB, serving as positive control), with six mice in each group. Each group of mice was numbered using picric acid. Five days after acclimatization, experiments began. Mice were anesthetized with ether and nasal instillation was initiated to establish the RSV infection model. Each mouse in the RSV infection group, RSV infection + low-dose BXM group, RSV infection + high-dose BXM group, and RSV infection + ribavirin positive control group received 100 μL of RSV virus stock solution. The wild-type group and BXM group received an equal volume of physiological saline. Nasal instillation was continued for 3 consecutive days. From day 4, intraperitoneal injections were administered for 3 consecutive days. Mice were treated on day 7. Mouse weight was measured at the same time each day, and their mental state, food intake, water intake, and activity status (ruffled fur, arched back, runny nose, cough, etc.) were closely observed.
[0046] ② Homogenize lung tissue and use TCID. 50Detect the viral load.
[0047] ③ The right lung of each mouse was removed, inflated, and fixed with 4% neutral buffered formaldehyde. The fixed tissue was embedded in paraffin and cut into 5 μm sections. The slides were then stained with hematoxylin and eosin (HE) and examined for histological changes under an optical microscope. Histopathological changes were assessed based on the degree of tissue inflammation, edema, and peribronchial inflammation.
[0048] ④ Lung tissue pathology score
[0049] The lung tissue HE pathological sections were scored according to the lung tissue pathological scoring system (as shown in Table 1). The total score = A + 3 × (B + C) + D + E, with a total score of 0-26 points.
[0050] Table 1. Lung tissue pathology scores
[0051]
[0052]
[0053] (2) To explore the molecular mechanisms related to the IKKβ / NF-κB pathway:
[0054] The expression of RSV F, p-IKKβ, p-NF-κB, and IL-6 proteins was detected by Western blot.
[0055] 3. Experimental Results
[0056] (1) Scanning electron microscopy confirmed the amplification of RSV in Hep-2 cells
[0057] Hep-2 cells are suitable for the culture and proliferation of various viruses. Therefore, the changes in Hep-2 cells after RSV infection were observed by scanning electron microscopy to assess whether the RSV-infected viral proliferation model was successfully established. Figure 3 The results showed: Figure 3 A represents a normal Hep-2 cell. Figure 3 B represents Hep-2 cells infected with RSV. Figure 3 In B, the formation of fusion lesions was observed in Hep-2 cells infected with RSV (indicated by the arrow), proving the success of the RSV-infected cell model.
[0058] (2) RSV infection of Hep-2 and BEAS-2B cells produces cytopathic effects
[0059] Hep-2 cells were used for RSV virus amplification, and BEAS-2B cells were used as the experimental subject; both were adherent cell lines. Microscopic observation of Hep-2 and BEAS-2B cells yielded the following results: Figure 4As shown, normal Hep-2 cells are irregularly polygonal ( Figure 4 A), while normal BEAS-2B cells are elongated spindle-shaped ( Figure 4 B). RSV infection of Hep-2 and BEAS-2B cells both produced a cytopathic effect (CPE). In Hep-2 cells, this manifested as cell rounding, detachment, aggregation, loss of cell boundaries, and cell membrane fusion. Figure 4 C); while BEAS-2B is characterized by elongated and stretched cells, with some cells becoming filamentous after being elongated, and widened intercellular spaces. Figure 4 D).
[0060] (3) Methylbardoxazoline inhibits RSV infection and the production of related inflammatory factors in BEAS-2B cells by inhibiting the IKKβ / NF-κB pathway.
[0061] To verify the cytotoxic effect of bardoxolone methyl (BXM) in BEAS-2B cells, BEAS-2B cells were treated with different concentrations of the drug for 72 hours. The results are as follows: Figure 5 As shown, Figure 5 Figure A shows the cytotoxicity of methylbardoxazoline in BEAS-2B cells and its inhibitory effect on RSV. Figure 5 B and Figure 5 C represents the graph showing BXM inhibiting RSV infection via the IKKβ / NF-κB pathway and the expression of related inflammatory factors resulting from infection. Figure 5 In this context, Cytoxicity refers to cytotoxicity, Inhibition refers to inhibition, Relative expression of mRNA refers to the relative expression of mRNA, and Relative protein expression refers to the relative expression of protein.
[0062] CCK8 assays revealed that when the concentration of methylbardoxolone was below 0.4 μM, the survival rate of BEAS-2B cells reached over 85%. However, when the concentration of methylbardoxolone was above 0.8 μM, microscopic observation showed varying degrees of shrinkage, deformation, and detachment of adherent cells. The CCK8 results were calculated. 50 ( Figure 5 A) Based on the safety range determined above, methylbardoxazoline at concentrations of 0.025, 0.05, 0.1, 0.2, and 0.3 μM was selected to determine the effects of different doses of methylbardoxazoline on the EC50 of RSV-infected BEAS-2B cells. 50 ( Figure 5A). RSV infection mediates multiple immune regulatory pathways, activating the NF-κB signaling pathway to produce a large number of inflammatory mediators, leading to repeated infections and exacerbating the disease process. Methylbardoxolone is an IKKβ inhibitor, therefore this invention explores whether methylbardoxolone can inhibit RSV infection by inhibiting the IKKβ / NF-κB pathway.
[0063] RT-PCR analysis revealed that RSV infection of BEAS-2B cells increased the expression of RSV, NF-κB, IKKβ, IL-6, and IL-8 mRNA 24 hours later. However, the expression of RSV, NF-κB, IKKβ, IL-6, and IL-8 mRNA decreased upon the addition of methylbardoxolone (*p<0.05, **p<0.01 indicates 500 TCID). 50 Compared with the normal control group, the RSV infection group # p < 0.05 ## p < 0.01 indicates 500 TCID 50 RSV+(0.1 / 0.3μM) methylbardoxazoline group with 500 TCID 50 Compared with the RSV infection group) Figure 5 B).
[0064] Western blot analysis revealed that RSV infection of BEAS-2B cells increased the expression of RSV F, IL-6, p-NF-κB, and p-IKKβ proteins. However, the expression of these proteins decreased upon the addition of methylbardoxazoline. This suggests that methylbardoxazoline may inhibit RSV infection and the expression of inflammatory factors by suppressing the IKKβ / NF-κB pathway (*p<0.05, **p<0.01 indicates 500 TCID). 50 Compared with the normal control group, the RSV infection group # p < 0.05 ## p < 0.01 indicates 500 TCID 50 RSV+(0.1 / 0.3μM) methylbardoxazoline group with 500 TCID 50 Compared with the RSV infection group) Figure 5 C).
[0065] (4) Effects of other IKKβ-specific inhibitors on BEAS-2B cells infected with RSV
[0066] A control experiment was conducted using the IKKβ-specific inhibitor TPCA-1 and methylbardoxolone, and BEAS-2B cells were treated for 24 hours.
[0067] The results showed that RSV mRNA expression was significantly increased in BEAS-2B cells after RSV infection.** P < 0.01), methylbadosolone, as an IKKβ inhibitor, can inhibit RSV mRNA expression ( ## P < 0.01 Figure 6 ,in Figure 6 The relative expression of mRNA (P>0.05) was observed, while the IKKβ-specific inhibitor TPCA-1 failed to inhibit RSV mRNA expression. These results indicate that not all IKKβ inhibitors can inhibit RSV mRNA expression.
[0068] (5) Changes in mouse body weight
[0069] SPF-grade BALB / c female mice were divided into groups according to... Figure 2 The mice were divided into six groups (wild-type group (untreated mice, blank control), RSV infection group, BXM (3 mg / Kg / d) group, RSV infection + low-dose BXM (1 mg / Kg / d) group, RSV infection + high-dose BXM (3 mg / Kg / d) group, and RSV infection + ribavirin (40 mg / Kg / d) positive control group) and treated accordingly. Mice were weighed at the same time each day, and the results are as follows: Figure 7 As shown, observations revealed that wild-type and BXM mice exhibited smooth fur, active behavior, and normal food and water intake. BXM mice initially showed a slight decrease in weight, which gradually stabilized, but their mental state, behavior, and diet remained largely unchanged. RSV-infected mice, after nasal drop experiments, all showed gradual weight loss, dry and disheveled fur, poor mental state, lethargy, and a tendency to huddle together, with reduced food and water intake. RSV-infected mice treated with low / high-dose BXM initially showed similar characteristics to the RSV-infected group, but from day 5 onwards, the RSV-infected mice treated with methylbadosol and ribavirin gradually increased in weight, and their abnormal mental and behavioral changes gradually improved. The most significant improvement was observed in the RSV-infected + high-dose BXM group (3 mg / kg / d), while the results for the RSV-infected + low-dose BXM group (1 mg / kg / d) and the RSV-infected + ribavirin group (40 mg / kg / d) were quite similar. This indicates that methylbadoxolone can inhibit RSV infection in mice, with the most significant effect observed at a BXM dose of 3 mg / kg / day. A BXM dose of 1 mg / kg / day showed an effect comparable to the positive control ribavirin. Figure 7 In this context, Virus refers to RSV infection, Drugs refers to administration of methylbardoxolone, Treat refers to treatment, and Weight of Mice refers to the weight of the mouse.
[0070] (6) Pathological changes and scoring of mouse lung tissue
[0071] SPF-grade BALB / c female mice were divided into groups according to... Figure 2 The experimental methods described above involved grouping (wild-type group, RSV infection group, BXM (3 mg / Kg / d) group, RSV infection + low-dose BXM (1 mg / Kg / d) group, RSV infection + high-dose BXM (3 mg / Kg / d) group, RSV infection + ribavirin (40 mg / Kg / d) positive control group) and treatment. Mice were sacrificed on day 7, and the lower lobe of the right lung was fixed in 4% paraformaldehyde for 48 hours and then stained with hematoxylin and eosin (HE). The results are as follows... Figure 8 As shown, where Figure 8 A represents the normal control group. Figure 8 Group B is the RSV infection group. Figure 8 C represents the RSV infection group plus low-dose BXM (1 mg / kg / d). Figure 8 Group D was the RSV infection group plus high-dose BXM (3 mg / kg / d). Figure 8 E represents the RSV-infected, ribavirin-positive control group (40 mg / kg / d). Figure 8 F represents the BXM group (3mg / Kg / d). The magnification of the upper layer image for each group is 200, and the magnification of the lower layer image is 400.
[0072] Figure 8 The results showed that the lung tissue and alveolar septa of mice in the wild-type group and the BXM group were normal. Figure 8 A, Figure 8 F). Compared with the wild-type group, the RSV-infected group showed significant inflammatory cell infiltration, increased nucleated cells, thickened alveolar septa, alveolar wall rupture and damage, inflammatory cell infiltration of bronchial small vessels, and bronchial exudation in the lung tissue. Figure 8 B). Compared with the RSV infection group, the RSV infection + low / high dose BXM group showed reduced inflammatory cell infiltration in lung tissue, intact alveolar walls, reduced alveolar septal thickening, and reduced bronchial small vessel inflammatory infiltration. The RSV infection + high dose BXM group (3mg / Kg / d) showed the most significant remission effect. Figure 8 D), while the RSV infection + low-dose BXM group (1 mg / Kg / d) ( Figure 8 C) Compared to the RSV infection + high-dose BXM group (3mg / Kg / d) Figure 8 D) The effect was not satisfactory. In the RSV infection + ribavirin positive control group (40 mg / kg / d), there was also a reduction in inflammation. Figure 8 E), but the effect was slightly worse than that of the RSV infection + high-dose BXM group (3mg / Kg / d).
[0073] In addition, lung tissue pathology scoring was performed (Table 1, Figure 9Statistical analysis showed that, similar to the RSV infection + low / high dose BXM group and the RSV infection + ribavirin positive control group, it significantly reduced lung tissue inflammation in mice (compared to the normal group, **p<0.01; compared to the infection group, the drug group, **p<0.01). ## p < 0.01), and the RSV infection + high-dose BXM group (3 mg / Kg / d) showed a more significant effect than the RSV infection + low-dose BXM group (1 mg / Kg / d). ▲▲ p < 0.01). Figure 9 The Histology score of lung tissue is the histological score of lung tissue.
[0074] (7) Viral load in mouse lung tissue
[0075] The changes in RSV viral titer in mouse lung tissue were further examined, and the viral titer in lung tissue was determined by the TCID50 method. Results showed that the RSV infection group had the highest RSV viral titer in lung tissue. Treatment with methylbadosolone and ribavirin significantly reduced the viral titer (the drug groups included: RSV infection + low-dose BXM (1 mg / kg / d), RSV infection + high-dose BXM (3 mg / kg / d), RSV infection + ribavirin (40 mg / kg / d) positive control group; comparisons were made between the drug groups and the RSV infection group). # p < 0.05 ## (p < 0.01) The viral titer in the RSV infection + high-dose BXM group (3 mg / Kg / d) decreased more significantly than that in the RSV infection + low-dose BXM group (1 mg / Kg / d) (**p < 0.01). Figure 10 (where Virus Titer is the viral titer).
[0076] (8) Methylbardoxazoline inhibits RSV infection and the production of related inflammatory factors in mice by inhibiting the IKKβ / NF-κB pathway.
[0077] After a virus invades the body, it produces a large number of inflammatory mediators, causing damage. In cell experiments, this invention has verified that methylbardoxolone can inhibit RSV infection and suppress the production of inflammatory factors by inhibiting the IKKβ / NF-κB pathway. Therefore, this invention further extracted mouse lung tissue from an RSV-infected mouse model and detected the expression of RSV F, p-IKKβ, p-NF-κB, and IL-6 proteins by Western blot. The results showed that the RSV F protein expression level in the RSV-infected group was higher than that in the wild-type group ( Figure 11A and 11C, **p<0.01); RSV infection + low-dose BXM (1mg / Kg / d) group and RSV infection + high-dose BXM (3mg / Kg / d) group significantly inhibited RSV F protein expression level ( Figure 11 A and 11C, ## p < 0.01), while the RSV infection + ribavirin (40 mg / Kg / d) positive control group showed a decrease in RSV F protein expression compared to the RSV infection group, but this was not statistically significant. Figure 11 A and 11C (p > 0.05) indicate that methylbardoxolone can also inhibit RSV infection in vivo. This invention further examined the expression of proteins related to the IKKβ / NF-κB pathway, and the results showed that compared with the wild-type group, the protein expression levels of p-IKKβ, p-NF-κB, and IL-6 were significantly increased in the RSV-infected group (p-IKKβ, p-NF-κB, and IL-6). Figure 11 A-11B, Figure 11 D-11F, *p<0.05, **p<0.01), the expression levels of p-IKKβ, p-NFκB, and IL-6 were consistent in the wild-type group and the BXM group. However, the expression levels of p-IKKβ, p-NF-κB, and IL-6 in the RSV infection + low-dose BXM (1 mg / Kg / d), RSV infection + high-dose BXM (3 mg / Kg / d), and RSV infection + ribavirin (40 mg / Kg / d) positive control group were significantly lower than those in the RSV infection group. Figure 11 A-11B, Figure 11 D-11F, # p < 0.05 ## p < 0.01. These results indicate that methylbardoxazoline inhibits RSV infection and the expression of related inflammatory factors in mice by suppressing the IKKβ / NF-κB pathway. Figure 11 In this context, "Relative protein expression" refers to the relative expression of proteins. Figure 11 In this context, "Relative protein expression" refers to the relative protein expression.
Claims
1. The use of methylbardosolone in the preparation of a drug for inhibiting respiratory syncytial virus infection, wherein the concentration of methylbardosolone is 0.01-20 mg / kg / d.
2. Application of methylbardoxolone in the preparation of drugs that inhibit respiratory syncytial virus infection by inhibiting the IKKβ / NF-κB pathway.
3. The application according to claim 2, characterized in that, The methylbardosorone can inhibit the expression of inflammatory factors in respiratory syncytial virus infection.
4. The application according to claim 2 or 3, characterized in that, The concentration of methylbardoxolone is 0.1-10 mg / kg / d.
5. The application according to claim 4, characterized in that, The concentration of methylbardoxolone is 1-5 mg / kg / d.
6. The application according to claim 5, characterized in that, The concentration of the methylbardoxolone is 1-3 mg / kg / d.
7. The application according to claim 6, characterized in that, The concentration of methylbardoxolone is 2.5-3 mg / kg / d.
8. The application according to claim 2 or 3, characterized in that, The dosage form of the drug is at least one of tablets, capsules, powders, granules, injections, and oral liquids.