Application of SRT2104 in the preparation of drugs for treating bronchial asthma

Abstract

The present invention discloses the use of SRT2104 in the preparation of a drug for treating bronchial asthma, and relates to the technical field of bronchial asthma treatment. The present invention comprehensively evaluates the activation effect of SRT2104 on airway epithelial SIRT1 and the relief effect on asthmatic airway inflammation under asthma conditions, confirms the effectiveness of SRT2104 in activating airway epithelial SIRT1 and controlling asthmatic airway inflammation under asthma conditions, and lays a foundation for the discovery of clinical medical targets and the development of drugs in the treatment of bronchial asthma.

Description

Technical Field

[0001] The present invention relates to the technical field of bronchial asthma treatment, and more specifically to the application of SRT2104 in the preparation of a medicament for treating bronchial asthma. Background Art

[0002] Bronchial asthma (abbreviated as asthma) is one of the most common respiratory diseases. It is a heterogeneous disease characterized by chronic airway inflammation and airway remodeling. It is characterized by multiple respiratory symptoms accompanied by reversible expiratory airflow limitation. Clinical manifestations include wheezing, shortness of breath, chest tightness and cough.

[0003] Airway epithelial cells play a core role in the pathogenesis of asthma. They are located between the body and the external environment and are the first line of defense against microorganisms, harmful gases and allergens. During the pathogenesis of asthma, endogenous and exogenous factors (biological, physical, chemical and allergic reactions, etc.) repeatedly stimulate the airway epithelium, causing epithelial damage. Once the airway epithelium is damaged, external allergens can easily enter the subcutaneous tissue and activate the immune system to trigger allergic reactions. More and more studies have found that the barrier function of airway epithelial cells mainly prevents the entry of harmful irritants through various intercellular connection mechanisms (such as tight junctions, adhesion junctions, etc.), and removes allergens, viruses and other external harmful factors through the mucociliary system and antimicrobial peptides. In healthy humans, airway epithelial cells maintain the dynamic balance of the airway environment through biochemical barriers, but when the airway barrier is damaged, the airway structure and related proteins will be damaged, leading to the occurrence and progression of asthma. Fungal and pollen extracts increase the barrier permeability of epithelial cells by affecting the expression of junction proteins. House dust mites destroy the barrier function of airway epithelial cells by activating the protease-activated receptor 2 (PAR-2) of airway epithelial cells. In addition, studies have found that viruses have a more significant effect on the barrier function of airway epithelial cells. Infected cells will undergo apoptosis or necrosis under the induction of the virus, thereby damaging the epithelial barrier. Many years ago, researchers have noticed that there are airway epithelial clots in the sputum of asthma patients, and the number of such epithelial cells increases significantly during attacks. Therefore, airway epithelial cell damage is considered to be a pathological feature of bronchial asthma and is closely related to airway responsiveness and disease severity. Silent information regulator 1 (SIRT1) is the most extensively and deeply studied member of the Sirtuins family. It can activate NAD +It senses changes in cellular energy metabolism and affects the acetylation level of substrates through its deacetylase activity, converting metabolic signals into epigenetic signals. It is currently known that SIRT1 can regulate more than 70 substrates, including p53, PGC-1α, NF-κB, FOXO, etc. SIRT1 interacts with different substrates to exert different functions. Therefore, SIRT1 can participate in the regulation of various cellular functions and physiological and pathological processes, for example, regulating cell damage repair growth, proliferation, apoptosis, autophagy and differentiation. It is widely involved in the body's inflammatory response, energy consumption, oxidative stress, aging, neural signals, and even circadian rhythms. Therefore, it can play an important role in a variety of diseases.

[0004] In recent years, SIRT1 has been considered as a protein molecule associated with asthma. The therapeutic potential of increased SIRT1 activity to relieve asthma has made this enzyme a very attractive target for drug design. SIRT1 regulators may be potential drugs for the treatment of asthma. However, it is still unknown whether SIRT1 is involved in the stress response of airway epithelial cells and inflammatory response-induced bronchial asthma. In other words, whether the expression of SIRT1 changes during airway epithelial cell damage and whether SIRT1 is expressed in airway epithelial cells to regulate the onset of bronchial asthma have not yet been reported.

[0005] SRT2104 is the first highly selective small molecule activator of SIRT1. Currently, our research on SRT2104 has entered the clinical transformation stage. Unfortunately, SRT2104 is currently basically used orally or intravenously. After entering the intestine, it is absorbed by the small intestinal epithelial cells and will produce a first-pass effect in the liver, which will affect the efficacy of SRT2104 and has poor targeting. Therefore, it is a technical problem that technicians in this field urgently need to solve to clarify whether SRT2104 can directly act on airway epithelial cells through other administration methods and explore whether it can be used as a new drug to treat bronchial asthma by acting on the SIRT1 target.

[0006] Further research has found that SRT2104 has good tolerance, and after being inhaled into the body through aerosolization, it has high targeting and acts specifically on airway epithelial cells, and no off-target effects or first-pass effects in the liver have been found. Moreover, the aerosol administration method is 1 / 4 to 1 / 10 of the dosage of intravenous administration, directly acts on airway epithelial cells, relieves bronchial asthma, and has a rapid onset of effect. Summary of the invention

[0007] In view of this, the present invention is proposed.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The application of SRT2104 in the preparation of a drug for treating bronchial asthma, SRT2104 enhances the deacetylation activity of SIRT1 on NF-κB p65.

[0010] Preferably, SRT2104 reduces the production of LPS-induced HBE inflammatory factors, including IL-25, TSLP, IL-33 and TNF-α.

[0011] Preferably, the intervention of SRT2104 reduced the infiltration of inflammatory cells in the BALF of asthmatic mice and reduced the inflammation score of lung tissue.

[0012] Preferably, SRT2104 reduces airway mucus secretion in asthmatic mice.

[0013] Preferably, SRT2104 is capable of increasing the expression level of SIRT1 in the lung tissue of asthmatic mice.

[0014] Through the above technical scheme, it can be known that compared with the prior art, the present invention uses HBE and SIRT1 gene knockout HBE cell lines to induce inflammatory response, and uses SRT2104 for treatment. By detecting the level of inflammatory factors, the degree of activation of SIRT1 and the acetylation level of NF-κB, it is clear that SRT2104 inhibits airway epithelial inflammation by activating SIRT1 and the anti-inflammatory mechanism. In addition, we successfully constructed an asthma model with wild-type mice, and used SRT2104 atomization treatment to observe the level of inflammatory factors in the alveolar lavage fluid of asthmatic mice, the level of inflammatory factors specific to the airway epithelium, the infiltration of inflammatory cells in the lung tissue and the secretion of airway mucus, and the expression level of SIRT1 in the lung tissue. Through the above experiments, the activation effect of SRT2104 on airway epithelial SIRT1 and the relief effect on asthmatic airway inflammation under asthma conditions were comprehensively evaluated, confirming the effectiveness of SRT2104 in activating airway epithelial SIRT1 under asthma conditions and controlling asthmatic airway inflammation.

[0015] Moreover, the direct administration of the drug through atomization in the present invention is different from the current oral or intravenous injection methods, which greatly reduces the dosage of the drug and quickly relieves the symptoms of asthma in a short period of time with almost no side effects. In addition, SRT2104 targets the SIRT1 protein in airway epithelial cells and achieves the effect of treating bronchial asthma by regulating the expression level of the protein, laying the foundation for the research and development of targeted drugs for the clinical treatment of bronchial asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 The attached figure shows the effect of SIRT1 gene knockout in HBE.

[0018] Figure 2 The attached figure shows the expression level of SIRT1 protein in HBE under the action of different concentrations of SRT2104; A: Western blot was used to detect the expression level of SIRT1 protein under the action of different concentrations of SRT2104. B: Tubulin was used as an internal reference, and the protein grayscale analysis method was used to statistically analyze the expression level of SIRT1 protein. Data are expressed as mean ± standard deviation, NS: P>0.05; ****: P<0.0001.

[0019] Figure 3 The attached figure shows the survival rate of HBE under the action of different concentrations of LPS.

[0020] Figure 4 The attached figure shows the expression levels of IL-33, IL-25 and TSLP in HBE under the action of different concentrations of LPS.

[0021] Figure 5 The attached figure shows that SRT2104 can effectively activate SIRT1 in LPS-induced inflammatory response.

[0022] Figure 6 The attached figure shows that SRT2104 can reduce the production of LPS-induced HBE inflammatory factors.

[0023] Figure 7 The attached figure shows that SRT2104 has no alleviating effect on the inflammatory response of HBE in which the SIRT1 gene was knocked out.

[0024] Figure 8 The attached figure shows that SRT2104 has no effect on the deacetylation activity of NF-κB p65 in HBE with knockout of SIRT1 gene.

[0025] Fig. 9 The attached figure shows the detection of inflammatory factors in plasma.

[0026] Fig.10 The attached figure shows that the intervention of SRT2104 reduced the infiltration of inflammatory cells in BALF of asthmatic mice.

[0027] Fig.11 The attached figure shows HE staining of lung tissue and inflammation score.

[0028] Fig.12 The attached figure shows PAS staining and inflammation score of lung tissue.

[0029] Fig.13 The attached figure shows the expression of SIRT1 around the mouse airway observed by IHC. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Experimental materials: HBE cells were provided by the Maternal and Child Research Institute of Shunde Women and Children's Hospital Affiliated to Guangdong Medical University. 6-8 week old SPF female C57BL / 6 mice weighing (20±4) g were purchased from Guangdong Medical Experimental Animal Center.

[0032] The main equipment is shown in Table 1;

[0033] Table 1

[0034]

[0035]

[0036] The main reagents are shown in Table 2;

[0037] Table 2

[0038]

[0039]

[0040] Configuration of main reagents

[0041] (1) TBST: 50 ml of 20× Tris buffered saline (TBS), add 949 ml of ultrapure water, then add 1 ml of Tween-20 solution, stir the buffer thoroughly until the liquid is clear, and store at room temperature.

[0042] (2) PBS buffer: Take 1 packet of PBS dry powder, add 2000 ml of pure water, stir thoroughly, and after the powder is completely dissolved, steam sterilize for 30 min and store in a refrigerator at 4°C.

[0043] (3) 5% skim milk blocking solution: weigh 5 mg skim milk powder, add 100 ml TBST solution, mix thoroughly, and store at -20°C.

[0044] (4) Goat serum blocking solution: add 9 ml PBS to 1 ml 10% goat serum, mix well, place in a 55°C water bath for 26 min, and store at -20°C.

[0045] (5) OVA suspension for intraperitoneal injection: Add 480 μg OVA and 24 mg Al(OH)3 to 2.4 ml normal saline, mix thoroughly and use immediately.

[0046] (6) 5% OVA nebulized solution: Weigh 5 g of OVA chicken egg albumin, add 100 ml of 0.9% saline, stir to mix, and store at 4°C.

[0047] (7) SRT2104 nebulizer solution: Weigh 4 mg of SRT2104, add 1 ml of DMSO, mix thoroughly, add 4 ml of PEG300, then add 1 ml of Tween, and finally add physiological saline to make up to 50 ml. Place in a 37°C water bath for 10 min and store at 4°C.

[0048] Example 1

[0049] Cultivation of HBE

[0050] (1) Recovery of HBE

[0051] ①Take out the DMEM complete medium from 4℃ and place it at room temperature for 30 minutes.

[0052] ②Turn on the water bath and adjust it to 37°C.

[0053] ③Take out the frozen cells and immediately place them in a water bath to thaw for 3 minutes.

[0054] ④ Transfer the cells to a centrifuge tube containing complete culture medium and centrifuge at 850 rpm for 4 minutes.

[0055] ⑤Discard the supernatant in the centrifuge tube, add complete culture medium, and use a pipette to gently blow away the cell clumps at the bottom of the centrifuge tube.

[0056] ⑥ Transfer the resuspended cells to a cell culture dish, shake slowly, let it stand for 15 minutes, then gently place the cell culture dish in a cell incubator (37°C, 5% CO2, 95% humidity) for culture.

[0057] (2) Passaging of HBE

[0058] ① Take out DMEM complete medium, 0.25% EDTA trypsin, and 1× PBS from 4°C and place them at room temperature for 30 minutes.

[0059] ②Discard the culture medium in the culture dish and wash the cells twice with 1×PBS.

[0060] ③ Add a small amount of 0.25% EDTA trypsin to the culture dish and place it in a cell incubator for digestion for 3 minutes.

[0061] ④ The cells flow like quicksand, indicating that the cells have been completely digested. Immediately add complete culture medium to terminate the digestion.

[0062] ⑤ Gently blow the culture medium several times to completely detach the cells from the culture dish.

[0063] ⑥ Transfer the cell suspension to a centrifuge tube and centrifuge at 850 rpm for 4 minutes.

[0064] ⑦ Refer to steps (1)-⑤-⑥.

[0065] (3) Cryopreservation of HBE

[0066] ①Observe under a microscope and wait until the cells grow to 90% density before freezing.

[0067] ② Refer to steps (2)-①-⑥.

[0068] ③Discard the supernatant in the centrifuge tube, add cell freezing solution, repeatedly blow the cell freezing solution to resuspend the cells in the freezing solution, and centrifuge the cells at 1×10 6 Transfer the cells to a cryopreservation tube at a density of 100 live cells / ml, seal the tube, affix a label with the cell information, and place the cryopreservation tube in a -80°C refrigerator. If the cells need to be stored for a long time, the cryopreservation tube can be transferred to a liquid nitrogen cabinet on the next day.

[0069] Example 2 Construction of HBE SIRT1 gene knockout cell line

[0070] (1) Determine the screening concentration of puromycin

[0071] ① Add HBE at 2×10 5 The cells were evenly plated at a density of 1.54 × 10 μl in a 12-well cell culture dish and placed in a cell incubator overnight.

[0072] ②Discard the culture medium in the culture dish and wash the cells twice with 1×PBS.

[0073] ③Add screening culture medium containing different concentrations of puromycin and culture in a cell incubator. (The concentrations of puromycin are: 0, 0.3, 0.5, 1, 2, 5, 10, 15, in μg / ml).

[0074] ④Observe cell growth after 48 hours and replace with fresh screening medium.

[0075] ⑤Observe and record cell survival every day, and use the lowest concentration that can effectively kill all cells after 72 hours of culture as the screening concentration of puromycin.

[0076] (2) Lentivirus infection of HBE

[0077] ① Resuscitate 293T packaging cells at 1×10 per well 6 The cells were evenly plated in a 6-well cell culture dish and placed in a cell incubator overnight.

[0078] ② When the cell density reaches 70%, transfect the recombinant plasmid PB-SIRT1-sgRNA into 293T cells.

[0079] ③ 48 hours after transfection, collect the cell supernatant and concentrate the collected supernatant according to the instructions of the lentivirus concentration kit.

[0080] ④ Resuscitate HBE cells at 1×10 5 The cells were evenly plated in a 12-well cell culture dish and cultured in a cell incubator.

[0081] ⑤ When the cell density reaches 60%, replace with fresh culture medium, infect HB E with the concentrated virus, add 1 μl / ml polybrene to increase the infection efficiency, and culture in a cell incubator.

[0082] ⑥ 8 hours after infection, discard the culture medium, wash the cells twice with 1×PBS, and replace with fresh culture medium.

[0083] ⑦ After 72 hours of infection, puromycin (0.5 μg / ml) was added for 3 days of screening. When the cells grew to a sufficient number, a portion of the cells was taken to identify the expression level of SIRT1.

[0084] (3) Western blot identification of SIRT1 protein expression level

[0085] ①Discard the culture medium, wash three times with 1×PBS, add protease inhibitors, place on ice for 40 min, collect cells with a cell scraper, transfer to a centrifuge tube, centrifuge (4°C 12000 rpm) for 15 min, and collect the supernatant.

[0086] ② Determine the protein concentration, divide the sample into several portions, and store them at -80℃.

[0087] ③ Prepare separation gel and concentration gel according to the instructions, and let it stand at room temperature for 30 minutes until the gel is completely solidified.

[0088] ④ Mix the protein sample and loading buffer in a ratio of 4 to 1, place in a boiling water bath for 13 minutes to denature the protein, and cool at room temperature for 10 minutes.

[0089] ⑤ Add samples to the gel wells in sequence and run the electrophoresis at a constant voltage of 80V for 30 minutes. When the bromophenol blue dye reaches the junction of the concentrated gel and the separation gel, adjust the voltage to 160V and stop the electrophoresis when the bromophenol blue dye reaches the end of the separation gel.

[0090] ⑥Cut the PVDF membrane according to the size of the gel, soak it in methanol for 2 minutes, separate the glass plate and take out the gel, make a transfer template in the order of "sponge + PVDF membrane + gel + sponge", place it in a rapid transfer instrument, and transfer quickly for 10 minutes.

[0091] ⑦ Take out the PVDF membrane, wash it twice with TBST, place it in 5% skim milk blocking solution, block it on a shaker at low speed for 50 minutes, and then wash it three times with TBST.

[0092] ⑧ Use antibody diluent to prepare SIRT1 anti-I dilution at a ratio of 1:900, add anti-I dilution and incubate on a shaker at 4°C overnight.

[0093] ⑨ Recover the diluted antibody I, wash the membrane with TBST for 5 times, 10 minutes each time, add antibody II diluted with 1% BSA solution, place on a shaker and incubate at low speed for 50 minutes, recover antibody II, and wash the membrane with TBST for 5 times, 8 minutes each time.

[0094] ⑩ Prepare ECT developer (A solution: B solution = 1:1) in a dark place, mix well and drop it onto the membrane surface, let it stand for 1 min, and place it in a gel imager for imaging. Tubulin was used as an internal reference, and the relative expression of the target protein was calculated using Image J software for statistical analysis.

[0095] The results show that Figure 1 Compared with wild-type HBE, the knockout group cells had no SIRT1 protein expression, suggesting that an HBE cell line with SIRT1 gene knockout was obtained.

[0096] Example 3 Western blot detection of different concentrations of SRT2104 on the expression level of SIRT1 protein

[0097] (1) Using DMSO as solvent, SRT2104 was prepared at concentrations of 1 μmol / L, 2 μmol / L, 4 μmol / L, and 8 μmol / L.

[0098] (2) Resuscitate HBE at 1×10 6 The cells were evenly plated in a 6-well cell culture dish and placed in a cell incubator overnight.

[0099] (3) When the cell density reaches 80%, fresh culture medium is replaced, and prepared SRT2104 of different concentrations is added, and the cells are cultured in a cell incubator.

[0100] (4) After culturing for 24 h, the culture medium was discarded, the cells were washed twice with 1× PBS, protease inhibitors were added, and the cells were placed on ice for 40 min. The cells were collected with a cell scraper and transferred to a centrifuge tube. The cells were centrifuged (4°C, 12,000 rpm) for 15 min, and the supernatant was collected.

[0101] (5) Refer to Example 3-(3)-②-⑩ steps.

[0102] The results showed that compared with the blank control group, the expression of SIRT1 protein was significantly upregulated when treated with 1μmol, 2μmol, and 4μmol of SRT2104 (P<0.0001), and the difference was statistically significant ( Figure 2 ). This suggests that SIRT1 can be activated when HBE is treated with 1μmol, 2μmol, and 4μmol of SRT2104. Therefore, we finally used 1μmol, 2μmol, and 4μmol of SRT2104 for subsequent experiments.

[0103] Example 4 LPS induces HBE inflammation

[0104] (1) CCK-8 method was used to detect the activity of HBE by different concentrations of LPS

[0105] ① Use sterile distilled water as the solvent to prepare LPS at concentrations of 2.5μg / ml, 5μg / ml, 10μg / ml, 15μg / ml, and 20μg / ml.

[0106] ② Resuscitation of HBE: Five experimental groups were set up according to the concentration of prepared LPS, with 6 replicate wells in each group. A blank control group was also set up. PBS buffer was added to the outermost wells of the 96-well plate to reduce the evaporation of the culture medium in the experimental group. 2×10 3 The cells were plated at an even density and placed in a cell incubator overnight.

[0107] ③ When the cell density reached 85%, the culture medium was discarded, and the five experimental groups were added with culture medium containing different concentrations of LPS, and the blank group was only added with the same amount of culture medium, and then placed in a cell incubator for 24 hours.

[0108] ④ Replace the culture medium with fresh one and add 10 μl of CCK-8 solution to each well. Be careful to avoid bubbles during the addition process. Place the 96-well plate with CCK-8 solution in a cell incubator and incubate for 1 hour.

[0109] ⑤ Take out the 96-well plate, avoid light, and use a microplate reader to detect the OD value of each well at a wavelength of 450nm. Cell survival rate = [(experimental well-blank well) / (control well-blank well)]*100%. Analyze and process the data and draw a curve.

[0110] The results showed that the survival rate of HBE in the 2.5μg / ml LPS group was (86.2±5.4)%, the survival rate of HBE in the 5μg / ml LPS group was (54.0±3.5)%, the survival rate of HBE in the 10μg / ml LPS group was (47.3±3.5)%, the survival rate of HBE in the 15μg / ml LPS group was (46.6±4.9)%, and the survival rate of HBE in the 20μg / ml LPS group was (36.5±1.2)%. This suggests that LPS can affect the survival of HBE, and the survival rate gradually decreases with the increase of LPS concentration ( Figure 3 )

[0111] (2) ELISA detection of the expression levels of HBE inflammatory factors by different concentrations of LPS

[0112] ① Resuscitate HBE, set up five experimental groups according to the concentration of prepared LPS, with 3 replicates in each group, and set up a blank control group at 1×10 6 The cells were evenly plated in a 6-well cell culture dish and cultured in a cell incubator.

[0113] ② When the cell density reached 80%, the culture medium was discarded and the cells were washed twice with 1×PBS. The culture medium containing different concentrations of LPS was added to the five experimental groups respectively, and the blank group was only added with the same amount of culture medium and cultured in a cell incubator for 24 hours.

[0114] ③ Collect the cell supernatant and centrifuge (3000 rpm, 15 min) to remove particles and polymers.

[0115] ④Take out the ELISA kit from 4℃ and equilibrate it at room temperature for 30 minutes.

[0116] ⑤ Take out the required strips from the aluminum foil bag of the kit, set up standard wells and sample wells, add 50μl of standards of different concentrations to the standard wells, add 40μl of sample diluent and then 10μl of the sample to be tested to the sample wells, and do not add anything to the blank wells.

[0117] ⑥Except for the blank wells, add 100 μl of HRP-labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C incubator for 1 hour.

[0118] ⑦ Carefully peel off the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 60 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 6 times.

[0119] ⑧ Add 50 μl of substrate A and B to each well and incubate in a 37°C incubator away from light for 20 min.

[0120] ⑨ Add 50 μl of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm using an enzyme-labeled instrument, calculate the concentration of inflammatory factors based on the standard curve, and draw a curve.

[0121] The results showed that compared with the blank control group, the expression levels of related inflammatory factors IL-33, IL-25, and TSLP in HBE showed a gradually increasing trend under the action of different concentrations of LPS, and in the 5μg / ml LPS group, the expression levels of IL-33, IL-25, and TSLP were significantly increased, P<0.0001, and the difference was statistically significant ( Figure 4 ). Therefore, we finally adopted 5 μg / ml LPS as the induction concentration for subsequent experiments.

[0122] Example 5 Effects of SRT2104 on LPS-induced HBE and SIRT1 gene knockout HBE

[0123] (1) Cell grouping

[0124] The cells were grouped and processed according to the following table:

[0125] Table 3 Cell grouping

[0126]

[0127] Figure caption: HBE-SIRT1 sgRNA: knockout of SIRT1 gene in airway epithelial cells

[0128] (2) Western blot detection of the expression levels of related proteins in LPS-induced HBE and SIRT1 knockout HBE by SRT2104

[0129] ① According to the grouping, culture medium containing LPS (5 μg / ml) was added for induction for 24 hours, and then culture medium containing different concentrations of SRT2104 was added and placed in a cell incubator for 24 hours.

[0130] ② Refer to Example 2-(3)-①-⑦ steps.

[0131] ③ Use antibody diluent to prepare the anti-I dilutions of SIRT1, NF-κb p65, and Ac-NF-κb p65 respectively. After adding the anti-I dilutions, place the cells on a shaker at 4°C and incubate overnight.

[0132] ④ Refer to Example 2-(3)-⑨-⑩ steps.

[0133] The results showed that compared with the blank control group, the SIRT1 protein in the LPS group was significantly downregulated, P<0.05, and the difference was statistically significant. The SIRT1 protein expression levels in the high, medium, and low dose groups of SRT2104 were gradually increased, P<0.001, and the difference was statistically significant ( Figure 5 ). This suggests that SRT2104 can effectively activate SIRT1 in LPS-induced inflammatory response in a dose-dependent manner.

[0134] (3) ELISA method was used to detect the effect of SRT2104 on LPS-induced HBE and related inflammatory factors in HBE with SIRT1 gene knockout

[0135] ① Culture HBE and HBE with SIRT1 knockout. When the cell density reaches 60%, discard the culture medium, wash three times with 1×PBS, add culture medium containing different concentrations of SRT2104 according to the grouping, culture for 24 hours, change the medium, add culture medium containing LPS (5 μg / ml), and place in a cell incubator for 24 hours.

[0136] ② Refer to Example 4-(2)-③-⑨ steps.

[0137] The results showed that for HBE cell lines, compared with the blank control group, the levels of IL-25, IL-33, and TSLP in the LPS group were significantly increased, P<0.05, and the difference was statistically significant. The levels of IL-25, IL-33, and TSLP in the SRT2104 treatment group showed a downward trend, among which IL-25 and TSLP in the SRT2104 treatment group decreased significantly, P<0.001, and the difference was statistically significant ( Figure 6 ).

[0138] For the cell lines with knockout of SIRT1 gene, the results showed that the levels of IL-25, TSLP, IL-6, and IL-33 in the high, medium, and low dose groups of SRT2104 were not significantly different from those in the LPS group, P>0.05( Figure 7 ). This suggests that SRT2104 has no effect on alleviating the inflammatory response of HBE with SIRT1 gene knockout.

[0139] At the same time, Western blot was used to detect the expression levels of NF-κB p65 and Ac-NF-κB p65 proteins. The results showed that there was no significant difference in the Ac-NF-κB p65 protein level between the high, medium and low dose groups of SRT2104 and the knockout control group and LPS group, indicating that SRT2104 had no effect on the deacetylation activity of NF-κB p65 in HBE with SIRT1 gene knockout ( Figure 8 ).

[0140] Example 6 Animal Experiment Grouping and Establishment of Asthma Model

[0141] (1) Animal grouping

[0142] ① Animal selection: SPF grade C57BL / 6 wild-type mice, female, 6 weeks old, weighing about 20 g.

[0143] ② Experimental grouping: 24 mice were randomly divided into four groups: PBS group, OVA group, OVA+solvent (DMSO) group, and OVA+SRT2104 group, with 6 mice in each group.

[0144] (2) Establishment of asthma model

[0145] ① The mice were sensitized by intraperitoneal injection of 40 μg OVA + 2 mg Al(OH)3 three times, once every 7 days on average, within 14 days.

[0146] ② On the 18th to 20th day, the mice were placed in a nebulizer box and given PBS, DMSO, and SRT2104 for three consecutive days according to the grouping.

[0147] ③ On the 21st to 23rd day, the mice were placed in a nebulizer, and 5% OVA was prepared with normal saline and nebulized for three consecutive days to stimulate asthma.

[0148] ④On the 24th day, the mice were killed and the specimens were collected.

[0149] Example 7 Detection of inflammatory factors in plasma

[0150] (1) Blood sampling from the eyeball

[0151] ① Anesthetize the mouse first. After ensuring that it is fully anesthetized, fix the mouse with one hand and cut off the mouse's whiskers to prevent blood contamination.

[0152] ② Gently press the skin of the eye on the blood-drawing side to make the eyeball congested and protrude.

[0153] ③ Use curved tweezers to quickly remove the eyeball and collect the blood flowing out of the eye socket. At the same time, use your left hand to gently press the mouse's heart to speed up the heart's pumping speed.

[0154] ④ Centrifuge the collected blood at 4°C, 2500 rpm for 15 min, and collect the plasma supernatant in a new test tube.

[0155] (2) ELISA to detect the expression level of inflammatory factors in plasma

[0156] ① Refer to Example 4-(2)-④-⑨ steps.

[0157] In this process, 24 C57BL / 6 wild-type mice were randomly divided into four groups: PBS group, OVA group, OVA+solvent (DMSO) group, and OVA+SRT2104 group, with 6 mice in each group. Asthmatic mice in the OVA+SRT2104 group were treated with 15 mg / kg of SRT2104 by aerosol. 24 hours after the last OVA challenge, blood was collected from the eyeballs, and the expression levels of IL-33, IL-25, and TNF-α in the plasma of mice in each group were detected by ELISA. The results showed that compared with the PBS group, the expression levels of IL-33, IL-25, and TNF-α in the OVA-induced group were significantly increased, P<0.0001, and the difference was statistically significant. There was no significant difference in the expression levels of IL-33, IL-25, and TNF-α between the OVA-induced group and the OVA+DMSO group, P>0.05. However, the expression levels of IL-33, IL-25, and TNF-α in the OVA+SRT2104 group were significantly decreased compared with the OVA group (P<0.01), and the difference was statistically significant ( Fig. 9 ).

[0158] Example 8 Detection of inflammatory factors in BALF

[0159] (1) Collection of BALF and inflammatory cells

[0160] ① After blood collection, mice were killed by cervical dislocation.

[0161] ② Open the chest, separate the mediastinum, draw 1 ml of normal saline with a syringe, and slowly push it into the lungs through the trachea. Repeat the lavage 3 times and then draw out the lavage fluid.

[0162] ③ The obtained lavage fluid was centrifuged at 2000 rpm at 4°C for 20 min, and the supernatant and precipitated cell components were collected separately.

[0163] (2) Inflammatory cell count in BALF

[0164] ① Add 1 ml of physiological saline to the obtained cell pellet and gently blow the cells to evenly distribute the cells to prepare a cell suspension.

[0165] ② Use an automated cell counting instrument to count the total number of cells in each group.

[0166] ③After counting, take 10μl of the cell suspension for smear. The smear should be of appropriate thickness and dry naturally for 24 hours.

[0167] ④ Add drops of Wright-Giemsa complex stain to cover the entire specimen smear and stain for 1 minute at room temperature.

[0168] ⑤ Add an equal amount of PBS (PH 6.4-6.8), gently shake the slide, mix thoroughly with the Wright-Giemsa complex stain, and stain at room temperature for 3 minutes.

[0169] ⑥ Rinse three times with nearly neutral water, blot dry, and examine under a microscope.

[0170] (3) ELISA detection of inflammatory factor expression levels in BALF

[0171] ① Refer to Example 4-(2)-④-⑨ steps.

[0172] The results showed that the total number of cells in BALF of OVA group and OVA+DMSO group was significantly higher than that of PBS group, P<0.0001, and the difference was statistically significant. The total number of cells in BALF of OVA+SRT2104 group was significantly lower than that of OVA group and OVA+DMSO group, P<0.001, and the difference was statistically significant. The results of Wright-Giemsa staining showed that the total number of inflammatory cells in BALF of OVA group and OVA+DMSO group was significantly higher than that of PBS group, and the total number of inflammatory cells in BALF of OVA+SRT2104 group was significantly lower than that of OVA group. The results of inflammatory factor detection in BALF supernatant showed that the expression levels of IL-33, IL-25, and TNF-α in OVA-induced group were significantly increased compared with PBS group, P<0.0001, and the difference was statistically significant. There was no significant difference in the expression levels of IL-33, IL-25, and TNF-α between OVA group and OVA+DMSO group, P>0.05. However, the expression levels of IL-33, IL-25, and TNF-α in the OVA+SRT2104 group were significantly decreased compared with the OVA group (P<0.05), and the difference was statistically significant ( Fig.10 ).

[0173] Example 9 Lung Histopathological Examination and Inflammatory Scores

[0174] (1) Lung tissue sections

[0175] ① Remove the left lung, taking care to ensure the integrity of the lung tissue and avoid damage.

[0176] ② Place the extracted lung tissue in an appropriate fixative (4% paraformaldehyde) to avoid deformation of the lung tissue.

[0177] ③ The tissue acquires rigidity through gradient dehydration treatment with ethanol of different concentrations.

[0178] ④ Soak the dehydrated specimen thoroughly in wax.

[0179] ⑤The specimen was embedded in wax and cut into thin slices with a thickness of 5 μm. All slices were controlled at the distal end of the airway for immunohistochemistry (IHC), periodic acid-Schiff staining (PAS staining), and hematoxylin-eosin staining (HE staining).

[0180] HE staining was used to observe the infiltration of inflammatory cells in lung tissue. The results showed that compared with the PBS group, a large number of inflammatory cells infiltrated around the trachea and blood vessels in the OVA group and the OVA+DMSO group. The inflammatory score was estimated by the size of the inflammatory infiltration area, P<0.0001, and the difference was statistically significant. In the OVA+SRT2104 group, the infiltration of inflammatory cells around the trachea and blood vessels was significantly reduced compared with the OVA group and the OVA+DMSO group. The inflammatory score was estimated by the size of the inflammatory infiltration area, P<0.001, and the difference was statistically significant ( Fig.11 )

[0181] (2) PAS staining

[0182] ① Place the slices in an oven at 60°C for 20 min.

[0183] ② Dewaxing: Place the sections in xylene I for 8 min, xylene II for 8 min, anhydrous ethanol for 5 min, 95% alcohol for 5 min, 80% alcohol for 5 min, 75% alcohol for 5 min, and TBST for 10 min.

[0184] ③ Soak the slices in PAS dye solution for 8 minutes. Take them out of the PAS dye solution and rinse them with running water for 10 minutes.

[0185] ④ Soak the slices in Schiff reagent for 30 minutes and rinse with running hot water for 3 minutes.

[0186] ⑤ Soak the slices in Hematoxylin reagent for 5 minutes and rinse with running water for 5 minutes.

[0187] ⑥ Add an appropriate amount of Bluing reagent to cover the slice, incubate for 1 minute, and rinse with running water for 2 minutes.

[0188] ⑦ Incubate the slices in Light green reagent for 3 minutes and soak them in distilled water for 1 minute.

[0189] ⑧ Rapid dehydration (favorable for the preservation of slices): Dehydrate the slices in anhydrous ethanol for 5 minutes, 95% alcohol for 5 minutes, 80% alcohol for 5 minutes, 75% alcohol for 5 minutes, xylene I for 2 minutes, and xylene II for 2 minutes.

[0190] ⑨ Sealing: Take the slices out of xylene and let them dry, add neutral gum, cover the slide, and gently press the slide to allow the neutral gum to quickly spread and cover the tissue. Move quickly to prevent the slices from drying. Examine under a microscope.

[0191] ⑩ PAS staining result interpretation: The airway mucus secretion capacity was evaluated based on the staining results: glycogen and polysaccharides were purple-red, and the cell nucleus was blue. 0 points: no inflammation was detected; 1 point: a small number of inflammatory cells were occasionally seen; 2 points: one to three inflammatory cells were seen around most bronchi or blood vessels; 3 points: four to five inflammatory cells were seen around most bronchi or blood vessels; 4 points: more than five inflammatory cells were seen around most bronchi or blood vessels. 3-5 fields of view were randomly selected from each slice, and the double-blind method was used for evaluation, and the average value of each group was used as the final result.

[0192] Compared with the PBS group, the mucus in the cytoplasm of airway epithelial cells in the OVA group and the OVA+DMSO group was greatly formed. The airway mucus secretion ability was evaluated according to the staining results, P<0.0001, and the difference was statistically significant. The synthesis of mucus in the cytoplasm of airway epithelial cells in the OVA+SRT2104 group was significantly reduced compared with the OVA group and the OVA+DMSO group. The airway mucus secretion ability was evaluated according to the staining results, P<0.0001, and the difference was statistically significant ( Fig.12 ).

[0193] (3) IHC dual color

[0194] ① Refer to steps (2)-①-②.

[0195] ② Antigen repair: Prepare antigen repair diluent with deionized water, place the slices in the antigen repair solution and boil in boiling water for 20 minutes. After natural cooling, place the slides in PBS (PH7.4) and wash on a decolorizing shaker 3 times, 5 minutes each time.

[0196] ③ Block endogenous peroxidase: Put the slices into 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes, put the slides in PBS (PH7.4) and wash them on a decolorizing shaker for 3 times, each time for 5 minutes.

[0197] ④ Blocking: Add 3% BSA in the tissue circle to evenly cover the tissue, and block at room temperature for 30 minutes.

[0198] ⑤ Add Antibody I: Gently shake off the blocking solution, prepare Antibody I, dilute Anti-SIRT1 at a ratio of 1:100, add 100μl of Antibody I on the slices, place the slices flat in a humidified box and incubate at 4°C overnight.

[0199] ⑥ Add antibody II: Place the slide in PBS (PH7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add 100 μl of antibody II (HRP labeled) in the circle to cover the tissue and incubate at room temperature for 60 minutes.

[0200] ⑦DAB color development: Place the slide in PBS (PH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, and control the color development time under a microscope. The positive color is brown-yellow, and the slices are rinsed with tap water to stop the color development.

[0201] ⑧Re-stain the cell nucleus: Re-stain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin bluing solution, and rinse with running water.

[0202] ⑨ Dehydration and sealing: Place the slices in 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal the slices.

[0203] ⑩Microscopic examination: Image analysis under a white light microscope

[0204] IHC staining was used to observe the expression of SIRT1 around the airways of each group of mice. The results showed that SIRT1 was highly expressed in the airway epithelium in the PBS group. Compared with the PBS group, the expression of SIRT1 in the airway epithelium of the OVA group and the OVA+DMSO group was significantly decreased. The expression of SIRT1 in the airway epithelium of the OVA+SRT2104 group was significantly increased compared with the OVA group and the OVA+DMSO group. This suggests that SIRT1 is highly expressed in the airway epithelium and decreases in asthma, and SRT2104 can increase the expression level of SIRT1 in the lung tissue of asthmatic mice ( Fig.13 ).

[0205] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0206] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of SRT2104 in the preparation of a drug for treating bronchial asthma, characterized in that: SRT2104 enhances the deacetylation activity of SIRT1 on NF-κB p65.

2. The use according to claim 1, characterized in that: SRT2104 reduces the production of LPS-induced HBE inflammatory factors, including IL-25, TSLP, IL-33, and TNF-α.

3. The use according to claim 1, characterized in that: Intervention with SRT2104 reduced inflammatory cell infiltration in BALF and lung tissue inflammation scores in asthmatic mice.

4. The use according to claim 1, characterized in that: SRT2104 reduces airway mucus secretion in asthmatic mice.

5. The use according to claim 1, characterized in that: SRT2104 can increase the expression level of SIRT1 in the lung tissue of asthmatic mice.

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