CN116473979A

CN116473979A - Application of methylbardoxolone in preparation of medicines for inhibiting respiratory syncytial virus infection

Info

Publication number: CN116473979A
Application number: CN202310407222.4A
Authority: CN
Inventors: 刘代顺; 龚玲; 王虹苹; 崔国祯; 韦涛; 徐杭; 刘悰跃
Original assignee: First People's Hospital Of Zunyi Third Affiliated Hospital Of Zunyi Medical College; Zhuhai Campus Of Zunyi Medical University
Current assignee: First People's Hospital Of Zunyi Third Affiliated Hospital Of Zunyi Medical College; Zhuhai Campus Of Zunyi Medical University
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-07-25

Abstract

The invention belongs to the technical field of medicines and provides application of methylbardoxolone in preparation of medicines for inhibiting respiratory syncytial virus infection, and the invention establishes RSV infected cells and a mouse model, and observes pathological changes effect of BEAS-2B cells after RSV infection by using an inverted microscope, and a CCK8 method detects cytotoxicity and antiviral effect of the methylbardoxolone, and detects mRNA expression of RSV, IL-6, IL-8, IKKK and NF- κB by reverse transcription polymerase chain reaction, and protein immunoblotting detects expression of RSV F, p-IKKK and p-NF- κ B, IL-6 protein, so that pharmacodynamics of the methylbardoxolone for relieving lung inflammation injury caused by RSV infection through an IKK beta/NF- κB channel is proved for the first time, and the methylbardoxolone can be used for preparing medicines for inhibiting respiratory syncytial virus infection.

Description

Application of methylbardoxolone in preparation of medicines for inhibiting respiratory syncytial virus infection

Technical Field

The invention relates to the technical field of medicines, in particular to an application of methylbardoxolone in preparing a medicine for inhibiting respiratory syncytial virus infection.

Background

Respiratory syncytial virus (Respiratory Syncytial Virus, abbreviated RSV) is one of the most common viral pathogens of the respiratory tract and is widely spread worldwide. RSV is the leading cause of premature infants and infants Mao Zhiyan and pneumonia, accounting for about 60% of all preschool children worldwide, estimated that 94600-149700 children under 5 years die of RSV infection worldwide. RSV is also an important pathogen for respiratory tract infections in elderly people over 65 years of age and in immunocompromised individuals. According to the statistics of world health organization, about 3300 thousands of infected persons are worldwide, wherein more than 340 ten thousand of severely infected persons need hospitalization, 10-31% of hospitalized patients need to enter an intensive care unit, and 3-17% need to be mechanically ventilated. RSV infection currently lacks a mature vaccine and effective therapeutic. Therefore, intensive research into drugs and mechanisms for treating RSV infection is particularly important. Early stage is found by clinical specimens and in vitro experiments: RSV infection mediates innate immune damage to Toll-like receptor 3 (TLR 3) and Toll-like receptor 7 (TLR 7), and by stimulating NF- κb signaling pathways, a large number of inflammatory mediators such as IL-6, IL-8, IL-10, IL-13, IL-32, TNF- α, etc. are produced, inflammatory factors are increased, and lung injury is exacerbated.

RSV has been found for over 60 years, but has very limited therapeutic options. To date, RSV control drugs certified by the united states food and drug administration (Food and Drug Administration, FDA) are only two: inhaled ribavirin and palivizumab. Ribavirin is a nucleoside analog, a broad-spectrum antiviral drug. Early studies showed that it rapidly cleared RSV infection, reduced patient symptoms and shortened hospital stay, but there are other perspectives that ribavirin did not reduce mortality after RSV infection and reduced mechanical ventilation time in critically ill patients. In addition, ribavirin causes toxic side effects of drugs such as hemolysis, congenital teratogenesis, cardiovascular toxicity, and the like, which makes its use more and more questioned. Palivizumab is a humanized mouse monoclonal antibody aiming at respiratory syncytial virus fusion protein (RSV F), is only suitable for preventing severe lower respiratory tract infection of children caused by RSV, can obviously reduce hospitalization times, hospitalization time and death rate, but needs to be re-injected every half year due to fast metabolism, is expensive, and is not marketed in China.

There is currently a lack of mature vaccines and effective therapies for RSV infection, and therefore there is a need to develop an effective method of treating RSV infection.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides application of the methylbardoxolone in preparing medicines for inhibiting respiratory syncytial virus infection, the invention detects cytotoxicity and antiviral effect of the methylbardoxolone through a CCK8 method by observing pathological effects after the BEAS-2B cells are infected with RSV through an inverted microscope, and detects mRNA expression of RSV, IL-6, IL-8, IKKKbeta and NF-kappa B through a reverse transcription polymerase chain reaction (RT-PCR), and protein immunoblotting (Western blot) detects the expression of RSV F, p-IKKbeta and p-NF-kappa B, IL-6 proteins, and the molecular mechanism of the methylbardoxolone for resisting the RSV infection is related to an IKKbeta/NF-kappa B channel, so that the methylbardoxolone is proved to inhibit respiratory syncytial virus infection and inhibit respiratory syncytial virus inflammatory factor expression through inhibiting the IKKbeta/NF-kappa B channel.

In a first aspect, the invention provides the use of methylbardoxolone in the manufacture of a medicament for inhibiting respiratory syncytial virus infection.

In particular to the application of the methylbardoxolone in preparing medicines for inhibiting respiratory syncytial virus infection.

Methyl bardoxolone (Bardoxolone Methyl, BXM for short) is a novel small molecular compound, is a semisynthetic triterpene oleanolic acid derivative, has CAS number of 218600-53-4, and has effects of resisting oxidative stress, inflammation, proliferation, and cancer. The direct acting targets of the methyl bardoxolone are Keap1 and IKKK beta, and the direct acting targets are combined with Keap1 to destroy cysteine residues, so that Nrf2 is released, and an antioxidant and anti-inflammatory reaction can occur when Nrf2 is released. Methylbardoxolone also binds directly to Cys-179 in the IKKKβ activation loop, inhibiting NF- κB activation, and thus inhibiting downstream pro-inflammatory pathways.

Use of methylbardoxolone for the manufacture of a medicament for inhibiting respiratory syncytial virus infection by inhibiting the ikkβ/NF- κb pathway.

Use of methylbardoxolone for the manufacture of a medicament for inhibiting expression of inflammatory factors of respiratory syncytial virus by inhibiting the ikkβ/NF- κb pathway.

Use of methylbadosolone for the manufacture of a medicament for inhibiting respiratory syncytial virus infection and expression of respiratory syncytial virus inflammatory factor by inhibiting the ikkβ/NF- κb pathway.

Preferably, the concentration of the methyl bardoxolone is 0.01-20mg/Kg/d.

Further preferably, the concentration of the methyl bardoxolone is 0.1-10mg/Kg/d.

Further preferably, the concentration of the methyl bardoxolone is 1-5mg/Kg/d.

More preferably, the concentration of the methyl bardoxolone is 1-3mg/Kg/d.

More preferably, the concentration of the methyl bardoxolone is 2.5-3mg/Kg/d.

Preferably, the dosage form of the medicine is at least one of tablets, capsules, powder, granules, injection and oral liquid.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention proposes that the methylbardoxolone can be used for preparing medicines for inhibiting respiratory syncytial virus infection, no related researches on the molecular mechanism of RSV infection treatment and inflammatory injury by the methylbardoxolone exist at present, through establishing RSV infection cells and a mouse model, observing the pathological changes effect of the RSV infected by BEAS-2B cells by an inverted microscope, detecting the cytotoxicity and antiviral effects of the methylbardoxolone by CCK8 reagent (Cell Counting Kit-8), detecting the mRNA expression of RSV, IL-6, IL-8, IKKKbeta and NF-kappa B by reverse transcription polymerase chain reaction (RT-PCR), detecting the expression of RSV F, p-IKKKbeta and p-NF-kappa B, IL-6 protein by Western blot, the invention firstly proves the pharmacodynamics of the methylbardoxolone for relieving the lung inflammatory injury caused by infection through the IKKKbeta/NF-kappa B pathway, and the methylbardoxolone can be used for preparing medicines for inhibiting respiratory syncytial virus infection so as to provide promising medicines, therapeutic targets and strategies for clinical diagnosis;

(2) The invention utilizes the methyl bardoxolone to prepare the medicine for inhibiting the infection of respiratory syncytial virus and the expression of inflammatory factors of the respiratory syncytial virus by inhibiting an IKKbeta/NF- κB pathway, and can regulate and control the treatment effect by controlling the concentration of the methyl bardoxolone, wherein the treatment effect of the methyl bardoxolone with the application amount of 1mg/Kg/d can reach the level equivalent to the ribavirin with the application amount of 40mg/Kg/d, and the treatment effect of the methyl bardoxolone with the application amount of 3mg/Kg/d on RSV infected mice is optimal.

Drawings

FIG. 1 is a schematic diagram of an experimental method of an in vitro experiment;

FIG. 2 is a schematic diagram of the grouping and technical route of in vivo experiments;

FIG. 3 is a diagram of RSV infected Hep-2 cells visualized by scanning electron microscopy;

FIG. 4 is a diagram of RSV infection Hep-2 and BEAS-2B cells observed by a microscope;

FIG. 5 is a graph of the inhibitory effect of methylbardoxolone on RSV by the IKKKβ/NF- κB pathway in BEAS-2B cells;

FIG. 6 is a graph showing the effect of TPCA-1 on RSV infection in BEAS-2B cells;

FIG. 7 is a graph showing the change in body weight of mice in different groups;

FIG. 8 is a hematoxylin-eosin staining (HE staining) plot of mouse lung tissue in different groups;

FIG. 9 is a graph showing lung tissue pathology scores of mice in different groups;

FIG. 10 is a graph showing the results of mouse lung tissue virus titer determinations in different groups;

FIG. 11 is a graph of the inhibition of RSV infection and associated inflammatory factor production by the inhibition of the IKKKβ/NF- κB pathway in mice by methylprednisolone.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.

The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.

1. Experimental method

1. Cell experiment:

with the aim of exploring whether the methylbardoxolone can inhibit RSV infection and whether the methylbardoxolone can inhibit inflammatory injury caused by RSV infection through an IKKbeta/NF-kappa B pathway, a RSV infection cell model is established, and the effect and molecular mechanism of the methylbardoxolone on resisting the RSV infection through the IKKbeta/NF-kappa B pathway are studied.

(1) BEAS-2B (human bronchial epithelial cells), hep-2 (human laryngeal carcinoma epithelial cells, RSV-amplified vector) cells were purchased from American Type Culture Collection (ATCC) and were obtained from 2 nd to 3 rd generation cell lines.

(2) Preparation of cellular RSV infection model:

BEAS-2B and Hep-2 cells were resuscitated and passaged, RSV expansion was performed in Hep-2 cells, and the amplified TCID was determined ₅₀ Then with 500TCID ₅₀ RSV infects BEAS-2B.

(3) Discussing the molecular mechanism associated with the IKKbeta/NF- κB pathway:

(1) BEAS-2B cells were used as subjects and were divided into the following groups according to FIG. 1: normal control group, 500TCID ₅₀ RSV infection group, 500TCID ₅₀ RSV+0.1. Mu.M Methylbardoxolone group, 500TCID ₅₀ Rsv+0.3 μm methyl bardoxolone group, 4 total groups. Observing the pathological effect of BEAS-2B cells infected with RSV by an inverted microscope; detecting cytotoxicity and antiviral effects of the methylbardoxolone by a CCK8 method; RSV, IL-6, IL-8, ikkβ and NF- κb mRNA expression were detected by reverse transcription polymerase chain reaction (RT-PCR); detection by Western blot (Western blot)Expression of RSV F, p-IKKK beta, p-NF- κ B, IL-6 proteins.

(2) The IKKbeta specific inhibitor TPCA-1 and the methyl bardoxolone are used for control experiments, and the experiments are divided into: normal control group, 500TCID ₅₀ RSV infection group, 500TCID ₅₀ RSV+0.3. Mu.M Methylbardoxolone group, 500TCID ₅₀ Rsv+0.3 mu MTPCA-1 group, detection of RSV mRNA expression by RT-PCR demonstrated that not all ikkβ inhibitors can inhibit RSV infection through the ikkβ/NF- κb pathway.

2. Animal experiment

(1) Preparation of mouse RSV infection model:

(1) healthy, female SPF-grade BALB/c mice (6-8 weeks old, body weight 20-25 g) were randomly divided into according to FIG. 2: wild type group (i.e., untreated mice, blank), RSV-infected group, BXM (3 mg/Kg/d) group, RSV-infected+low dose BXM (1 mg/Kg/d) group, RSV-infected+high dose BXM (3 mg/Kg/d) group, RSV-infected+ribavirin (40 mg/Kg/d) positive control group (ribavirin RIB, as positive control), 6 total groups of 6 mice each, each group of mice was numbered with picric acid. The experiment was started after 5 days of adaptive feeding, nasal injection was started after the mice were anesthetized with diethyl ether, 100 μl of RSV virus stock solution was instilled into each of the RSV infection group, RSV infection+low dose BXM group, RSV infection+high dose BXM group, RSV infection+ribavirin Lin Yangxing control group, equal volumes of physiological saline were instilled into the wild type group and BXM group, nasal injection was continued for 3 days, and the mice were treated on day 7 after 3 days of continuous intraperitoneal injection. The weight of the mice was measured at the same time every day, and the mental, feeding, drinking and activity states (hair frying, back bow, nasal discharge, cough, etc.) of the mice were closely observed.

(2) Lung tissue homogenate using TCID ₅₀ The viral load was detected.

(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 HE and examined for histological changes by light microscopy, and the histopathological changes were assessed according to the extent of tissue inflammation, edema and peribronchial inflammation.

(4) Lung tissue pathology scoring

Lung tissue HE pathological sections were scored according to the lung tissue pathology scoring system (as in table 1), total score = a+3× (b+c) +d+e, total score 0-26.

TABLE 1 pulmonary pathology score

(2) Discussing the molecular mechanism associated with the IKKbeta/NF- κB pathway:

the expression of RSV F, p-IKKK beta and p-NF-kappa B, IL-6 proteins was detected by Western blot.

3. Experimental results

(1) Scanning electron microscopy confirmed that RSV was amplified in Hep-2 cells

Hep-2 cells are suitable for the culture and proliferation of a variety of viruses. Therefore, changes after RSV infection of Hep-2 cells were observed by scanning electron microscopy to assess whether the virus proliferation model after RSV infection was successfully constructed (fig. 3). The results show that: FIG. 3A shows normal Hep-2 cells, FIG. 3B shows Hep-2 cells after RSV infection, and FIG. 3B shows the formation of fusion lesions (indicated by arrows) in the Hep-2 cells after RSV infection, demonstrating success of the RSV infected cell model.

(2) Production of cytopathic effects upon RSV infection of Hep-2 and BEAS-2B cells

Hep-2 cells are cells required for the expansion of RSV virus, BEAS-2B cells are subjects, and both cells are cells grown on the wall. Hep-2 and BEAS-2B cells were observed by a microscope, and as a result, as shown in FIG. 4, normal Hep-2 cells were irregularly polygonal (FIG. 4A), while normal BEAS-2B cells were long-fusiform (FIG. 4B). Both Hep-2 and BEAS-2B cells were infected with RSV, which produced cytopathic effects (Cytopathic Effect, CPE), wherein Hep-2 exhibited cell rounding, shedding, aggregation, etc., and part of the cell boundaries disappeared, and cell membranes fused (fig. 4C); BEAS-2B, in turn, is characterized by elongated and stretched cells, and by partial elongation of the cells into filaments, the cell-cell gap is widened (FIG. 4D).

(3) Inhibition of RSV infection and related inflammatory factor production by the inhibition of the IKKKbeta/NF- κB pathway in BEAS-2B cells by methylbardoxolone

To verify the toxic effect of methylbardoxolone (Bardoxolone Methyl, BXM) on BEAS-2B cells, BEAS-2B cells were treated with different concentrations of drug for 72 hours, respectively. The results are shown in FIG. 5, wherein FIG. 5A shows cytotoxicity and RSV inhibition effect of methylbardoxolone in BEAS-2B cells, and FIGS. 5B and 5C show schematic representation of BXM inhibiting RSV infection and related inflammatory factor expression caused by the infection via IKKKβ/NF- κB pathway, respectively. In FIG. 5, cytotoxicity is cytotoxicity, inhibition is Inhibition, relative expression of mRNA is relative expression of mRNA, and Relative protein expression is relative protein expression.

CCK8 detection shows that when the concentration of the methyl bardoxolone is below 0.4 mu M, the survival rate of BEAS-2B cells reaches more than 85 percent, and when the concentration of the methyl bardoxolone is above 0.8 mu M, the attached cells are observed to shrink, deform and fall off to different degrees under a microscope, and CC is calculated ₅₀ (FIG. 5A), based on the safety margin measured above, 0.025, 0.05, 0.1, 0.2 and 0.3. Mu.M of Methylbadosol were selected to determine the EC of varying doses of Methylbadosol on RSV infected BEAS-2B cells ₅₀ (FIG. 5A). RSV infection mediates multiple immunomodulatory pathways, producing a large number of inflammatory mediators by activating the NF- κb signaling pathway, causing recurrent infections to exacerbate disease progression, whereas methylbardoxolone is an ikkβ inhibitor, and the present invention discusses whether methylbardoxolone can inhibit RSV infection by inhibiting the ikkβ/NF- κb pathway.

The RT-PCR detection shows that the expression of RSV, NF- κ B, IKK beta, IL-6 and IL-8mRNA is increased after 24 hours of RSV infection of BEAS-2B cells, but the expression of RSV, NF- κ B, IKK beta, IL-6 and IL-8mRNA is reduced when the methyl bardoxolone is added (p < 0.05, p < 0.01 is 500 TCID) ₅₀ The RSV infected group was compared to the normal control group, ^# p＜0.05， ^## p < 0.01 is 500TCID ₅₀ RSV+ (0.1/0.3. Mu.M) methyl bardoxolone group and 500TCID ₅₀ RSV infected group) (fig. 5B).

Through Western blot detection, after the BEAS-2B cells are infected by RSV, the expression of RSV F, IL-6, p-NF- κB and p-IKKKβ proteins is increased, but when methyl bardoxolone is added, the expression of RSV F, IL-6, p-NF- κB and p-IKKKKβ proteins is reduced. It is demonstrated that the effect of inhibiting RSV infection and expression of inflammatory factors (p < 0.05; p < 0.01 is 500 TCID) can be achieved by inhibiting IKKK beta/NF- κB pathway ₅₀ The RSV infected group was compared to the normal control group, ^# p＜0.05， ^## p < 0.01 is 500TCID ₅₀ RSV+ (0.1/0.3. Mu.M) methyl bardoxolone group with 500TCID ₅₀ RSV infected group) (fig. 5C).

(4) Effect of other ikkβ -specific inhibitors on BEAS-2B cells after RSV infection

BEAS-2B cells were treated with the IKKKbeta specific inhibitor TPCA-1 in a control experiment with methyl bardoxolone for 24 hours.

The results show that: after infection of BEAS-2B cells with RSV, the expression of RSV mRNA was significantly increased ^** P is less than 0.01), and the methylbardoxolone can inhibit the expression of RSV mRNA as an IKKbeta inhibitor ^## P < 0.01) (FIG. 6, where Relative expression of mRNA in FIG. 6 is the relative expression of mRNA), whereas the IKKKβ -specific inhibitor TPCA-1 was unable to inhibit RSV mRNA expression (P > 0.05). The above results indicate that not all ikkβ inhibitors can inhibit RSV mRNA expression.

(5) Weight change in mice

SPF-grade BALB/c female mice were grouped (6 groups total, wild-type group (i.e., mice without any treatment, blank), RSV-infected group, BXM (3 mg/Kg/d) group, RSV-infected+low dose BXM (1 mg/Kg/d) group, RSV-infected+high dose BXM (3 mg/Kg/d) group, RSV-infected+ribavirin (40 mg/Kg/d) positive control group) and treated as per the method of FIG. 2. The mice were weighed at the same time every day, and as shown in fig. 7, it was observed that the wild type mice Mao Seshun and the BXM mice were smooth, active, fed and drunk normally, and the BXM mice initially had slightly reduced body weight and then gradually maintained stable body weight, but had no significant change in mental behavior and diet. After the RSV infected mice are subjected to nasal drip experiments, the weight of the mice is gradually reduced, the hair is dry and disordered, the spirit is poor, the mice are inactive and happy and piled, and the feed intake and the water intake are reduced. RSV infected + low/high dose BXM group mice initially performed similarly to the RSV infected group, but from day 5 on, the mice in the RSV infected group treated with methylbardoxolone and ribavirin had progressively higher body weight and progressively improved mental performance, with the most significant improvement in the RSV infected + high dose BXM group (3 mg/Kg/d) and the RSV infected + low dose BXM group (1 mg/Kg/d) being closer to the RSV infected + ribavirin group (40 mg/Kg/d). It is demonstrated that the methylbardoxolone inhibits RSV infection in mice, and the effect is most remarkable when BXM is dosed at 3mg/Kg/d, and the effect can reach the level equivalent to that of positive control ribavirin when BXM is dosed at 1 mg/Kg/d. In FIG. 7, virus is RSV infection, drugs is administered methyl bardoxolone, treat is treatment, weight of Mice.

(6) Mouse lung tissue pathological changes and scoring

SPF-grade BALB/c female mice were grouped (wild type, RSV infected, BXM (3 mg/Kg/d), RSV infected+low dose BXM (1 mg/Kg/d), RSV infected+high dose BXM (3 mg/Kg/d), RSV infected+ribavirin (40 mg/Kg/d) positive control group) and treated as the experimental method in FIG. 2. Mice were sacrificed on day 7, and the lower right lung lobes of the mice were fixed with 4% paraformaldehyde for 48h and then HE stained, as shown in fig. 8, where fig. 8A is a normal control group, fig. 8B is an RSV-infected group, fig. 8C is an RSV-infected+low dose BXM group (1 mg/Kg/D), fig. 8D is an RSV-infected+high dose BXM group (3 mg/Kg/D), fig. 8E is an RSV-infected+ribavirin Lin Yangxing control group (40 mg/Kg/D), fig. 8F is a BXM group (3 mg/Kg/D), the upper panel magnification is 200, and the lower panel magnification is 400.

The results of fig. 8 show: the lung tissue and alveolar septum of wild type and BXM group mice were normal (fig. 8A, 8F). In the RSV-infected group, compared with the wild-type group, the lung tissue of the RSV-infected group had significant inflammatory cell infiltration, nucleated cell increase, alveolar space thickening, alveolar wall fracture injury, bronchi small vessel inflammatory cell infiltration, bronchi concomitant exudation (fig. 8B). In contrast, the RSV infection+low/high dose BXM group (fig. 8D) showed the most remarkable remission effect in the RSV infection+high dose BXM group (3 mg/Kg/D) compared to the RSV infection group (fig. 8C) due to the reduction of inflammatory cell infiltration in lung tissue, intact alveolar wall, reduced alveolar septum thickening, and reduced inflammatory infiltration in bronchi, whereas the RSV infection+low dose BXM group (1 mg/Kg/D) showed less excellent remission effect in the RSV infection+high dose BXM group (3 mg/Kg/D) (fig. 8D). There was also an inflammatory remission (FIG. 8E) in the RSV infection+ribavirin Lin Yangxing control group (40 mg/Kg/d), but slightly less effective than the RSV infection+high dose BXM group (3 mg/Kg/d).

In addition, lung histopathological scores were also performed (Table 1, FIG. 9), and it was found that the RSV infection+low/high dose BXM group and the RSV infection+ribavirin Lin Yangxing control group were similar in that they significantly reduced lung tissue inflammation (infection group compared with normal group, p < 0.01; drug group compared with infection group, ^## p < 0.01), and the effect of RSV infection + high dose BXM group (3 mg/Kg/d) is more obvious than that of RSV infection + high dose BXM group (1 mg/Kg/d) ^▲▲ p < 0.01). Wherein Histology score of lung tissue in fig. 9 is the histological scoring of lung tissue.

(7) Mouse lung tissue viral load

Further, the change of the RSV titer in the lung tissue of the mice was detected, and the virus titer of the lung tissue was measured by the TCID50 method. The results show that: the RSV titer of the lung tissue in the RSV infected group is highest, the virus titer is obviously reduced after the drug treatment of the methylbardoxolone and the ribavirin (the drug group comprises the RSV infection + low dose BXM (1 mg/Kg/d) group, the RSV infection + high dose BXM (3 mg/Kg/d) group, the RSV infection + ribavirin (40 mg/Kg/d) positive control group, the drug group is compared with the RSV infected group, ^# p＜0.05， ^## p < 0.01), the viral Titer was more pronounced (p < 0.01) in RSV infection + high dose BXM group (3 mg/Kg/d) than in RSV infection + low dose BXM group (1 mg/Kg/d) (fig. 10, where Virus Titer is the viral Titer).

(8) Methylbadoxolone inhibits RSV infection and related inflammatory factor production in mice by inhibiting the IKKKbeta/NF- κB pathway

The present invention has verified in cellular experiments that methylbardoxolone inhibits RSV infection and inhibits inflammatory factor production by inhibiting the ikkβ/NF- κb pathway after viral entry into the body resulting in a large amount of inflammatory mediators that cause damage to the body. Thus, the inventionFurther extracting mouse lung tissues from an RSV infected mouse model, and detecting expression conditions of RSV F, p-IKKK beta, p-NF- κB and IL-6 protein by Western blot. The results show that: RSV F protein expression levels were elevated in RSV-infected groups compared to wild-type groups (fig. 11A and 11C, < p < 0.01); RSV infection + low dose BXM (1 mg/Kg/d) and RSV infection + high dose BXM (3 mg/Kg/d) significantly inhibited RSV F protein expression levels (figures 11A and 11C, ^## p < 0.01), while the positive control group with RSV infection plus ribavirin (40 mg/Kg/d) showed a decrease in RSV F protein expression compared to the RSV-infected group, but was not statistically significant (FIGS. 11A and 11C, p > 0.05), indicating that the methylbardoxolone also inhibited RSV infection in vivo. The invention further detects the expression of the IKKbeta/NF- κB pathway related protein, and the result shows that: the protein expression levels of p-IKKbeta, p-NF- κB and IL-6 were significantly increased in the RSV-infected group compared to the wild-type group (FIGS. 11A-11B, 11D-11F, p < 0.05, p < 0.01), the protein expression levels of p-IKKbeta, p-NF- κB and IL-6 were consistent in the wild-type group and BXM group, but were significantly decreased in the post-infection RSV-infected+low dose BXM (1 mg/Kg/D), RSV-infected+high dose BXM (3 mg/Kg/D) and RSV-infected+ribavirin (40 mg/Kg/D) positive control groups (FIGS. 11A-11B, 11D-11F), ^# p＜0.05， ^## p < 0.01). The above results demonstrate that methylbardoxolone achieves an effect of inhibiting RSV infection and related inflammatory factor expression in mice by inhibiting the ikkβ/NF- κb pathway (Relative protein expression is relative protein expression in fig. 11). Wherein Relative protein expression in FIG. 11 is relative protein expression.

Claims

1. Use of methylbardoxolone in the manufacture of a medicament for inhibiting infection by respiratory syncytial virus.

2. Use of methylbardoxolone for the manufacture of a medicament for inhibiting respiratory syncytial virus infection by inhibiting the ikkβ/NF- κb pathway.

3. Use of methylbardoxolone for the manufacture of a medicament for inhibiting expression of inflammatory factors of respiratory syncytial virus by inhibiting the ikkβ/NF- κb pathway.

4. Use of methylbadosolone for the manufacture of a medicament for inhibiting respiratory syncytial virus infection and expression of respiratory syncytial virus inflammatory factor by inhibiting the ikkβ/NF- κb pathway.

5. The use according to claim 4, wherein the concentration of the methylbardoxolone is 0.01-20mg/Kg/d.

6. The use according to claim 5, wherein the concentration of the methylbardoxolone is 0.1-10mg/Kg/d.

7. The use according to claim 6, wherein the concentration of the methylbardoxolone is 1-5mg/Kg/d.

8. Use according to claim 7, characterized in that the concentration of the methylbardoxolone is 1-3mg/Kg/d.

9. Use according to claim 8, characterized in that the concentration of the methylbardoxolone is 2.5-3mg/Kg/d.

10. The use according to claim 4, wherein the pharmaceutical formulation is at least one of a tablet, a capsule, a powder, a granule, an injection, and an oral liquid.