New application of tripterine in anti-inflammatory treatment of severe influenza A virus infection

Abstract

The invention discloses a novel application of tripterine in anti-inflammatory treatment of severe influenza A virus infection. The invention relates to a novel application of tripterine in anti-inflammatory treatment of severe influenza A virus infection, and discloses that tripterine can effectively inhibit inflammatory response caused by severe influenza A virus infection and reduce pathological injury of lungs without affecting antiviral immune response.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to a new use of tripterine in anti-inflammatory treatment of severe infection of influenza A virus. BACKGROUND

[0002] Influenza A Virus (IAV) causes about 3-5 million severe cases and hundreds of thousands of deaths worldwide each year, of which more than 80% of the death cases are in high-risk groups such as the elderly, children, pregnant women and patients with chronic diseases. With the acceleration of global aging and the continuous increase of people with metabolic diseases and immune deficiency, the severity of influenza A has gradually posed a major challenge to global public health. Although antiviral drugs such as neuraminidase inhibitors (e.g. oseltamivir) can effectively inhibit IAV replication, their efficacy is highly dependent on the "golden 48-hour" window period, and the frequent occurrence of drug-resistant strains further limits the clinical benefits. More seriously, severe patients are often accompanied by "inflammatory storm" caused by excessive activation of the host immune system, and single antiviral treatment is difficult to reverse lung immunopathological damage. Therefore, it is of great significance to study the lung immune damage / protection mechanism and explore possible immune intervention strategies to improve the clinical cure rate of severe influenza.

[0003] Existing anti-inflammatory strategies have limitations. On the one hand, due to the dynamic superposition of different effector targets (neutrophil extracellular traps (NETs), inflammasome, etc.) in the process of severity, there is a lack of unified regulation hub, and the new anti-inflammatory drugs based on single target (tocilizumab, baricitinib) at this stage have limited clinical efficacy because they cannot cover multiple damage pathways. On the other hand, although glucocorticoids are a broad-spectrum anti-inflammatory solution, they may increase the risk of bleeding and secondary infection, and there is controversy in clinical use, and the "Influenza Diagnosis and Treatment Scheme (2025 Edition)" also does not recommend routine use. SUMMARY

[0004] The present application provides a new use of tripterine in anti-inflammatory treatment of severe infection of influenza A virus, which solves the aforementioned problems, and the specific scheme is as follows:

[0005] The new use of tripterine in anti-inflammatory treatment of severe infection of influenza A virus.

[0006] Further, the drug can reduce lung pathological damage, inhibit the formation of neutrophil extracellular traps, and improve the survival rate of the host.

[0007] Further, the drug inhibits the formation of neutrophil extracellular traps by down-regulating the Clcn3 gene, thereby significantly regulating the dysregulated immune response induced by influenza A virus.

[0008] Further, the drug is a combination of celadixine and the antiviral drug maraviroc.

[0009] Further, the dosage form of the drug is oral preparation, injection, inhalation or suppository.

[0010] Further, the oral preparation is tablet, capsule or syrup; the injection is intravenous injection or intramuscular injection; the inhalation is dry powder inhalation or atomization inhalation.

[0011] Further, the drug further comprises at least one pharmaceutically acceptable excipient selected from fillers, binders, disintegrants, lubricants, stabilizers, preservatives or flavorings.

[0012] Further, the preparation method of the drug comprises mixing celadixine with at least one pharmaceutically acceptable excipient to form the required dosage form.

[0013] Further, the preparation method of the drug comprises mixing celadixine with maraviroc, and then mixing with at least one pharmaceutically acceptable excipient to form the required dosage form.

[0014] Further, the drug is used for preventing or treating severe infection caused by influenza A virus, including but not limited to acute respiratory distress syndrome, pulmonary fibrosis or multiple organ failure.

[0015] The present application provides a new use of a drug that can effectively inhibit the inflammatory response caused by severe infection of influenza A virus, reduce lung pathological damage and does not affect the antiviral immune response, i.e. the use of celadixine in the preparation of a drug for treating severe infection of influenza A virus. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments or prior art of the present application, the drawings needed in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0017] Figure 1 The results of different treatment groups in the model of severe infection of influenza A virus are shown in the following table, including survival curve, body weight curve, lung gross pathology examination and inflammatory factor detection. P<0.05 is significant, with statistical significance, P less than 0.01 is extremely significant, with extremely high statistical significance.

[0018] Figure 2The lung tissue pathological examination results of different treatment groups, including H&E staining, Masson staining, and frozen section staining results, wherein P<0.05 is significant, with statistical significance, P less than 0.01 is extremely significant, with extremely high statistical significance. Figure 2 The lung tissue pathological examination results of different treatment groups, including H&E staining, Masson staining, and frozen section staining results, wherein P<0.05 is significant, with statistical significance, P less than 0.01 is extremely significant, with extremely high statistical significance.

[0019] Figure 3 The experimental results of Celastrol inhibiting the generation of NETs by inhibiting the expression of Clcn3 in neutrophils, wherein P<0.05 is significant, with statistical significance, P less than 0.01 is extremely significant, with extremely high statistical significance. DETAILED DESCRIPTION

[0020] The present application will be specifically introduced below in combination with the drawings and specific examples.

[0021] The technical solutions and effects of the present application will be further illustrated by experimental data below.

[0022] Experiment one:

[0023] DMSO, Baloxavir marboxil (BA), Celastrol (Cel), and Baloxavir marboxil combined with Celastrol (BA+Cel) were used to treat severe influenza A virus infection models, respectively. The treatment effect of Celastrol on severe influenza A virus infection was evaluated by drawing survival curves, body weight curves, lung gross pathology examination, and inflammatory factor detection.

[0024] Specifically, the mice were anesthetized by intraperitoneal injection of Avertin, and then infected with 100 plaque-forming units (PFU) of virus through the nose, with a total volume of 40 μl. Control mice received an equal volume of normal saline. Virus titers (PFU) were determined by plaque assay on MDCK cells by serial dilution of virus solution. After infection, mice were monitored daily for weight loss and survival, for a maximum of 14 days or until clinical endpoints.

[0025] Control mice received an equal volume of PBS. Baloxavir marboxil (1985606-14-1, MCE) was administered orally by gavage once a day at a dose of 10 mg / kg. Celastrol (34157-83-0, MCE) was administered intraperitoneally once every two days at a dose of 1 mg / kg. Control mice received an equal volume of PBS.

[0026] In addition, the health status of mice throughout the experiment was assessed using a clinical scoring system. Mice were considered to have reached a clinical endpoint when they lost more than 25% of their initial body weight. The clinical scoring criteria were as follows: (1 point each) piloerection, hunched posture, partially closed eyes, labored breathing, decreased activity, moving only when touched; or (2 points for more severe manifestations).

[0027] Survival curve analysis: As shown in Figure 1 (A), survival analysis found that emodin combined with maraviroc can significantly improve the survival rate of mice, higher than maraviroc and emodin alone.

[0028] Body weight curve analysis: As shown in Figure 1 (B), by drawing the body weight curve, it was found that emodin combined with maraviroc can make mice recover faster.

[0029] Lung macroscopic pathology examination: Animal model establishment, 6-8 weeks old SPF C57BL / 6 mice were selected, and intranasal inoculation of influenza A virus (IAV, H1N1 subtype, 100 PFU / each) was used to establish a severe infection model. Experimental grouping: randomly divided into four groups (n=8), control group (DMSO group): 48h after infection, intraperitoneal injection of DMSO dissolved in normal saline (0.1mL / 10g); maraviroc single drug group: 48h after infection, oral maraviroc (10mg / kg) once a day; emodin single drug group: 48h after infection, intraperitoneal injection of emodin (1mg / kg) every 48h; combination therapy group: maraviroc and emodin were administered according to the above dose and time sequence. Sampling and processing: after 5 days of continuous administration, the mice were sacrificed under carbon dioxide anesthesia, and the double lung tissue was completely stripped under sterile conditions and rinsed in pre-cooled normal saline, followed by gross observation and subsequent pathological analysis.

[0030] The following visual assessment was performed on fresh lung tissue:

[0031] Bleeding point area: count the red bleeding spots on the surface of the lung as a percentage of the total lung area (<25%: mild; 25-50%: moderate; >50%: severe);

[0032] Consolidation area: assess the area of atelectasis or hardening of the lung lobe due to inflammatory exudation (<25%: mild; 25-50%: moderate; >50%: severe);

[0033] Color and texture: normal is white sponge-like, congestion / edema is dark red and tough in texture, and consolidation is light pink.

[0034] Gross examination of the lung was performed, as shown in Figure 1(C) shows that the combination therapy group has less hemorrhagic spots and only mild consolidation (<10% area) at the edge of the lung lobe; the maraviroc monotherapy group has about 50% more hemorrhagic spots than the control group, but no significant difference in consolidation area; the triptolide monotherapy group has <10% immune hemorrhagic spots, but moderate to severe consolidation (50-70% area) in the whole lung lobe; the DMSO control group has diffuse hemorrhagic spots with multifocal consolidation (>60% area) in the whole lung.

[0035] Mice treated with triptolide in combination with maraviroc have fewer hemorrhagic spots and lung consolidation. Surprisingly, mice treated with maraviroc alone show more hemorrhagic spots. Although the triptolide group has less hemorrhage, it shows significant pathological changes of lung consolidation.

[0036] Inflammatory factor detection: According to the above lung gross pathology examination experiment, grouping and drug administration were performed, and bronchoalveolar lavage fluid (BALF) was collected 5 days after drug administration. After the mice were anesthetized, the trachea was exposed and a venous indwelling needle (22G) was inserted; 0.8 mL of pre-cooled sterile PBS (37°C preheated, pH 7.4) was injected in several times, and gentle repeated aspiration was performed 3 times; repeated lavage was performed 3 times, and the recovered lavage fluid was combined (total recovery rate > 85%); centrifugation at 1000xg for 10 min at 4°C, separation of supernatant and aliquot to EP tube, -80°C frozen for standby. Using (ABplex Mouse Cytokine 7-Plex Assay Kit [RK04780], using The xMAP multi-factor detection system was used to detect 6 cytokines such as CXCL-1 and G-CSF.

[0037] As shown in Figure 1 (D), it was found that mice treated with triptolide in combination with maraviroc had lower levels of specified inflammatory factors by detecting inflammatory factors.

[0038] Experiment two: lung tissue damage and neutrophil extracellular trap network detection analysis of different treatment groups in severe influenza virus infection model

[0039] According to the above lung gross pathology examination experiment, grouping and drug administration were performed, and lung tissue was collected 5 days after drug administration.

[0040] Tissue pretreatment:

[0041] Fixation: After dissection, the middle lobe tissue of the lung was taken out and immediately rinsed with pre-cooled PBS (4°C) for 3 times, and immersed in 10% neutral buffered formalin (Sigma, HT501128) for 24 hours.

[0042] Dehydration and embedding: Gradient ethanol dehydration (70%→80%→90%→100% ethanol, 1.5 h each); xylene clearing twice (30 min each); wax immersion (60 °C paraffin wax, 3 times, 1 h each); embedding as paraffin blocks, section thickness 5 pm.

[0043] H&E staining procedure:

[0044] De-waxing and hydration: Xylene I, II 10 min each→100% ethanol x 2 times (5 min each)→gradient ethanol (95%→85%→70%, 3 min each)→distilled water immersion.

[0045] Hematoxylin staining: Harris hematoxylin staining solution (Solarbio, G1080) immersion staining 5 min→running water rinse 10 min to return blue.

[0046] Differentiation and eosin counterstaining: Acidic ethanol (1% HC + 70% ethanol) differentiation 3 s→running water rinse 15 min→eosin staining solution (Sigma, HT110232) counterstaining 2 min.

[0047] Dehydration and mounting: Gradient ethanol dehydration (70%→85%→95%→100%, 30 s each)→xylene clearing twice (5 min each)→neutral resin mounting.

[0048] Masson’s trichrome staining procedure:

[0049] De-waxing to water: Same as H&E staining procedure.

[0050] Nuclear staining: Weigert’s iron hematoxylin staining solution (Sigma, HT1079) staining 10 min→running water rinse 10 min.

[0051] Plasma cell differentiation: 1% hydrochloric acid ethanol differentiation 30 s→running water rinse to return blue.

[0052] Collagen fiber staining: Ponceau red-acid fuchsin mixture (0.7% Ponceau red + 1% acid fuchsin) staining 10 min→1% phosphomolybdic acid treatment 5 min (to remove non-specific staining).

[0053] Myofiber and cytoplasm staining: 2% aniline blue staining solution staining 3 min→1% glacial acetic acid differentiation 30 s.

[0054] Dehydration and mounting: Gradient ethanol dehydration→xylene clearing→neutral resin mounting.

[0055] NETs frozen section staining:

[0056] Tissue snap freezing and embedding: snap freezing step, take fresh lung tissue, immediately immerse in pre-cooled PBS (4℃) for 3 times (10 s / time) to remove surface bloodstains; place in the center of OCT embedding agent (Sakura 4583), quickly put into pre-cooled isopentane (-50℃ to -40℃) for 1 min (avoid ice crystal formation).

[0057] Section operation: cryostat microtome (Leica CM1950), box temperature -20℃, sample head temperature -18℃, cut into 8μm thickness.

[0058] Multiple immunofluorescence staining: fixation, the section is restored to room temperature, 4% paraformaldehyde (Beyotime P0099) room temperature fixation for 15 min; PBS washing 3 times (5 min / time). Permeabilization, 0.1% Triton X-100 (Sigma T8787) treatment for 10 min→PBS washing 3 times. Blocking, drop 5% bovine serum albumin (BSA, Servicebio G5001) blocking solution, 37℃ wet box incubation for 1 h. Primary antibody mixture preparation, rabbit anti-CHIT3 (Abeam ab5103, 1:200), rat anti-Ly6G (Abeam ab25377, 1:100) diluted in 1% BSA / PBS solution, mix well. Incubation conditions, drop the primary antibody mixture to cover the tissue area, 4℃ incubation in a wet box overnight (12-16 h); PBS washing 3 times, 5 min / time. Secondary antibody incubation, Cy3 labeled goat anti-rabbit (Beyotime A0516, 1:500), FITC labeled goat anti-rat (Preteintec SA00003-11, 1:500) 37℃ incubation for 1 h→PBS washing 3 times. Nucleus staining, DAPI solution (Sigma D9542, 1μg / mL) drop staining for 10 min→deionized water washing 3 times. Mounting and storage, drop ProLong Diamond antifade mounting medium (Invitrogen P36965), cover glass mounting, avoid light, stand for 24 h to make the mounting medium fully solidified;

[0059] As shown in Figure 2 (A), HE staining found that the immune cell infiltration of the combination group was reduced, and the interstitial inflammation of the lung was lighter. Masson staining found that the fibrosis degree of the combination group was reduced.

[0060] As shown in Figure 2 (B), according to the results of Figure 2 (A), the lung pathological damage was quantitatively analyzed by Smith score, and the results showed that the lung damage of the combination group was lower.

[0061] As shown in Figure 2 (C), the lung pathological damage was quantitatively analyzed by Smith score, and the results showed that the lung damage of the combination group was lower.Figure 2 (A) Quantitative analysis of blue fibrosis level after Masson staining showed that the lung fibrosis degree of the combination group was lower.

[0062] As shown in Figure 2 (D), the lung tissues of mice in different treatment groups after severe infection of influenza A virus were collected and subjected to frozen section and neutrophil extracellular trap detection analysis. The combination group can significantly reduce the generation of neutrophil extracellular traps.

[0063] As shown in Figure 2 (E), the CHiT3 level in (D) was analyzed by Image J software. The results showed that the combination therapy can reduce the generation of neutrophil extracellular traps. Figure 2 (D) The neutrophil marker protein Ly6G in (D) was analyzed, and the results showed that the combination therapy can reduce neutrophil infiltration.

[0064] As shown in Figure 2 (F), the CHiT3 level in (D) was analyzed by Image J software. The results showed that the combination therapy can reduce the generation of neutrophil extracellular traps. Figure 3 (D) The neutrophil marker protein Ly6G in (D) was analyzed, and the results showed that the combination therapy can reduce neutrophil infiltration.

[0065] Experiment three: tripterine inhibits the generation of neutrophil extracellular traps by down-regulating Clcn3 in neutrophils

[0066] Treatment and RNA extraction experimental grouping (n = 3 repeats per group): control group, untreated neutrophils; LPS group, 1 μg / mL LPS (Sigma L2630) stimulation for 6 h; LPS + tripterine group: 10 μM tripterine (MCE 34157-83-0)

[0067] Pretreatment for 1 h → LPS stimulation.

[0068] RNA extraction: TRIzol reagent (Invitrogen 15596018) was used, 1 × 10 6 Add 1 mL TRIzol to the cells; after chloroform layering, centrifuge (12,000 × g, 15 min, 4℃), precipitate RNA with isopropanol, dissolve with DEPC water; RNA purity detection (Nanodrop, A260 / 280≥1.8).

[0069] Reverse transcription and PCR amplification: cDNA synthesis, PrimeScript RT Kit (Takara RR037A), total RNA 1 μg; 20 μL system: 42℃ for 30 min → 85℃ for 5 s inactivation. Primer design: Clcn3 (Gene ID: 1182):

[0070] Forward 5'-GGAGGCAGCATTAACAGTTCT-3', Reverse 5'-TCGCACCCAATCAATAGTATG GA-3' (amplification fragment 102 bp); Reference gene GAPDH: Forward 5'-GGAGCGAGATCCCTCCAAAAT-3' Reverse 5'-GGCTGTTGTCATACTTCTTGG-3' (amplification fragment 197 bp). qRT-PCR reaction: SYBR Green Premix (Takara RR420A), 20 μL system: 95 °C 30 s→ 95 °C 5 s→ 60 °C 34 s (40 cycles), melting curve analysis; Sample cycle threshold (Ct value) calculation ΔΔCt, Clcn3 expression = 2^(-ΔΔCt).

[0071] Data analysis standardization: specificity verification: single peak confirmation by melting curve, no primer dimer; repeatability control: 3 technical repeats per sample, Ct value standard deviation ≤0.5.

[0072] Immunofluorescence detection of the inhibitory effect of Clcn3 knockdown on NETs:

[0073] Cell treatment and transfection: experimental grouping (n=4 per group); control group: neutrophils (untreated); LPS group: 1 μg / mL LPS stimulation for 4 h; LPS+siClcn3 group: Clcn3 siRNA (Santa Cruz sc-42381) transfection for 24 h→LPS stimulation.

[0074] Transfection method: Lipofectamine 3000 (Invitrogen L3000001), siRNA concentration 50 nM; knockdown efficiency was detected 24 h after transfection (Western blot verification, protein level decreased by ≥70%).

[0075] NETs fluorescent staining: cell fixation and permeabilization, 4% PFA room temperature fixation for 20 min → PBS wash 3 times; 0.1% Triton X-100 permeabilization for 10 min → PBS wash; blocking: 5% BSA room temperature for 1 h. Primary antibody mixture preparation, rabbit anti-CHIT3 (Abeam ab5103, 1:200), rat anti-Ly6G (Abeam ab25377, 1:100) diluted in 1% BSA / PBS solution, mix well. Incubation conditions, drop the primary antibody mixture to cover the cell sample, 4°C incubation in a wet box overnight (12-16 h); PBS wash 3 times, 5 min / time. Secondary antibody incubation, Cy3 labeled goat anti-rabbit (Beyotime A0516, 1:500), FITC labeled goat anti-rat (Preteintec SA00003-11, 1:500) 37°C incubation for 1 h in the dark → PBS wash 3 times. Nuclei staining, DAPI solution (Sigma D9542, 1 μg / mL) drop staining for 10 min → rinse with deionized water 3 times.

[0076] As shown in Figure 3 (A), it is proved by q-RT-PCR experiment that emodin inhibits the expression of Clcn3 under LPS induction.

[0077] As shown in ​ (B), it is proved by immunofluorescence that knocking down Clcn3 can inhibit the activation of neutrophil extracellular trap under LPS induction.

[0078] In summary, the present application provides a new use of emodin as a drug for reducing the inflammatory response of severe influenza virus infection. Emodin combined with maraviroc can improve the survival rate of mice with severe influenza virus infection and accelerate weight recovery, effectively reduce the level of inflammatory factors, prevent immune damage, reduce lung pathological damage and fibrosis level, and reduce the generation of neutrophil extracellular traps. Experimental data support the application prospect of emodin as an anti-inflammatory treatment for severe influenza virus infection, which can effectively improve the survival rate of the host and reduce lung pathological damage.

[0079] In the embodiments of the present application, the drug can reduce lung pathological damage, inhibit the generation of neutrophil extracellular traps, and improve the survival rate of the host.

[0080] In the embodiments of the present application, the drug inhibits the generation of neutrophil extracellular traps by down-regulating the Clcn3 gene, thereby significantly regulating the dysregulated immune response triggered by influenza virus A.

[0081] In the embodiments of the present application, the drug is a combination of emodin and the antiviral drug maraviroc.

[0082] In embodiments of the present application, the dosage form of the drug is an oral preparation, an injection, an inhalation or a suppository.

[0083] In embodiments of the present application, the oral preparation is a tablet, a capsule or a syrup; the injection is an intravenous injection or a muscle injection; the inhalation is a dry powder inhalation or a nebulized inhalation.

[0084] In embodiments of the present application, the drug further comprises at least one pharmaceutically acceptable excipient selected from a filler, a binder, a disintegrant, a lubricant, a stabilizer, a preservative or a flavoring agent.

[0085] In embodiments of the present application, the preparation method of the drug comprises mixing celastrol with at least one pharmaceutically acceptable excipient to form a desired dosage form.

[0086] In embodiments of the present application, the preparation method of the drug comprises mixing celastrol with maraviroc, and then mixing with at least one pharmaceutically acceptable excipient to form a desired dosage form.

[0087] In embodiments of the present application, the drug is used for preventing or treating severe infection caused by influenza A virus, including but not limited to acute respiratory distress syndrome, pulmonary fibrosis or multiple organ failure.

[0088] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the above embodiments do not limit the present application in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present application.

Claims

1. A new anti-inflammatory use of tripterygium wilfordii in severe influenza A virus infection.

2. The use according to claim 1, characterized in that The drug can reduce lung pathological damage, inhibit the generation of neutrophil extracellular traps, and improve host survival rate.

3. The use according to claim 2, characterized in that The drug inhibits the production of neutrophil extracellular traps by downregulating the Clcn3 gene, thereby significantly regulating the dysregulated immune response triggered by influenza A virus.

4. The use according to claim 1, characterized in that The drug is a combination of tripterine and the antiviral drug mabaloxavir.

5. The use according to claim 1, characterized in that The dosage form of the medicine is oral preparation, injection, inhalation or suppository.

6. The use according to claim 5, characterized in that ， According to the use of claim 4, the oral preparation is a tablet, capsule or syrup; the injection is an intravenous injection or an intramuscular injection; and the inhaler is a dry powder inhaler or a nebulized inhaler.

7. The use according to claim 1, characterized in that The drug further comprises at least one pharmaceutically acceptable excipient, which is selected from fillers, binders, disintegrants, lubricants, stabilizers, preservatives or flavoring agents.

8. The use according to claim 7, characterized in that The preparation method of the medicine comprises mixing tripterygium wilfordii with at least one pharmaceutically acceptable excipient to prepare a desired dosage form.

9. The use according to claim 7, characterized in that The preparation method of the medicine comprises mixing tripterine with mabaloxavir, and then mixing with at least one pharmaceutically acceptable excipient to prepare a desired dosage form.

10. The use according to claim 1, characterized in that The drug is used to prevent or treat severe infection caused by influenza A virus, including but not limited to acute respiratory distress syndrome, pulmonary fibrosis or multiple organ failure.

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