Home Medicinal chemistry Efficacy of treatment combining salmeterol and magnesium isoglycyrrhizinate in rats with chronic obstructive pulmonary disease

Efficacy of treatment combining salmeterol and magnesium isoglycyrrhizinate in rats with chronic obstructive pulmonary disease

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Preparation of MgIG and other drugs

MgIG injection was purchased from Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Lianyungang, China). The main chemical composition of this product is MgIG, with the molecular formula of C42H60MgO164 hours2O. Sal (CAS: 89365-50-4) was purchased from LGM Pharma with a purity of 99% and a molecular formula of C25H37NOPE4. Salmeterol xinafoate and fluticasone propionate powder for inhalation (50 μg: 250 μg) were purchased from Glaxo Operations UK Limited. All drugs were stored at 4°C before use.

COPD and MgIG treatment rat model

Male Wistar rats (body weight, 180 ± 20 g; age, 6-8 weeks) were supplied by Charles River Laboratory Animal Technology Co., Ltd. (Pinghu, China) (Certificate No. SCXK (Zhe) 2019e001). Experimental procedures involving the use of animals were in accordance with the guidelines for animal experimentation of Jiangsu Vocational College of Medicine. The protocol was approved by the Animal Ethics Committee of Jiangsu Vocational College of Medicine. All animals were reared under standard environmental conditions and fed a standard laboratory diet and tap water ad libitum.

COPD model in rats was established by smoking and endotracheal atomization (ETA) of lipopolysaccharide (LPS) endotoxin according to previous reports20. The rats were randomly divided into groups (10 rats in each group): control group (CON), COPD model group (MDL), salmeterol xinafoate group (Sal), MgIG group (MgIG), salmeterol xinafoate group and MgIG (Sal/MgIG), salmeterol xinafoate group and fluticasone propionate (Sal/Flu). With the exception of the CON group, rats in the other five groups were anesthetized with isoflurane gas on days 1 and 15, followed by sensitization with 100 μL of 1 mg/mL LPS by endotracheal spray using liquid aerosols (MicroSprayer® Aerosolizer, model IA-1B, Penn-Century, Inc. Wyndmoor, USA), respectively. These rats were also passively exposed to 5% (v/v) cigarette smoke in several fumigation boxes for 30 min twice with an interval of 6 h per day, from the 2nd to the 14th day. Cigarette smoke was produced by Hademen cigarettes (each contains 10 mg of tar, 1.0 mg of nicotine, and 12 mg of carbon monoxide) (Jinan, China). As the COPD model rat was obtained, the rats were challenged with cigarette smoke alone daily from day 16 to day 45 with the above method. In addition, the Sal group received 0.40 mg/kg/day (w/bw/day) of salmeterol xinafoate dissolved in normal saline, the MgIG group received 0.40 mg/kg/day (w/ bw/day) of MgIG, the Sal/Flu group received 0.40 mg/kg/day (w/bw/day) of salmeterol xinafoate powder and fluticasone propionate dissolved in normal saline, and the group Sal/MgIG received 0.40 mg/kg/day (w/bw/day) salmeterol xinafoate and 0.40 mg/kg/day (w/bw/day) MgIG by endotracheal spray 1 h before treatment of cigarette smoke on days 30 to 45, respectively, while the MDL group received 100 μL of 0.9% normal saline with the same method of administration. Rats in the CON group were sensitized and challenged with 100 μL of normal saline in the same manner and exposed to normal air. After 45 days, the rats were sacrificed with an intraperitoneal injection of sodium pentobarbital (150 mg/kg; Sigma-Aldrich; Merck KGaA). All methods were conducted according to ARRIVE guidelines (https://arriveguidelines.org).

Pulmonary function measurement

Rat lung function was assessed using the AniRes2005 lung function analysis system (Beilanbo Technology Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Briefly, after 45 days of treatment, rats were anesthetized with 3% sodium pentobarbital (70 mg/kg; Sigma-Aldrich; Merck KGaA) and placed in a fixed supine position in a capacity box, to which a airway tube connected to ventilator and signal conditioner, transmitting airflow and change data to computer. Then, endotracheal intubation was performed on the rats. Breath rate and expiration/inspiration time ratio have been preset to 75/min and 1.5:1, respectively16. The forced vital capacity (FVC) and the forced expiratory volume in 0.3 s (FEV0.3) were detected, and the ratio of the two (FEV0.3/FVC) was used as an index to assess lung function in rats.

Classification of cells in bronchoalveolar lavage fluid (BALF)

To assess airway inflammation, inflammatory cells accumulated in bronchoalveolar lavage fluid (BALF) were analyzed16. Briefly, the tracheas of anesthetized rats were surgically exposed and intubated. The lungs were washed with 3 sequences of 1 ml of Hank’s balanced salt solution without calcium or magnesium, and BALF was harvested and centrifuged (400g) at 4°C for 10 min. The total number of cells was counted with a standard hemocytometer after resuspending the cell pellet in 1 ml of Hank’s balanced salt solution. Another aliquot of cell resuspensions was used to prepare cell slides, followed by staining of cells with Liu’s staining solution (PS0290, Beijing Jin Ming Biotechnology Co., Ltd. China). According to standard morphological criteria, approximately 200 cells were counted and classified as macrophages, lymphocytes, neutrophils and eosinophils.

Measurement of inflammatory factors

To detect inflammatory factors, serum from rats of all groups was respectively collected. Levels of inflammatory factors, interleukin (IL)-6, interleukin (IL)-1β and tumor necrosis factor (TNF)-α, were determined at 1:40 dilution with ELISA kits (Beyotime, Shanghai, China ) following the manufacturer’s protocol.

Histopathological analysis

To assess histopathological changes in lung tissue, left upper lobe tissue was removed from anesthetized rats to prepare paraformaldehyde-fixed, paraffin-embedded tissue specimens with the usual method. The sample was cut into 4 μm thick pieces and stained with hematoxylin and eosin (H&E) according to standard protocols. Histopathological changes were observed under the light microscope. We randomly selected three visual fields (200×) from each H&E stained section and then divided the number of alveoli in each visual field by the area of ​​the visual field to obtain MAN (mean alveolar number). Finally, bronchial wall thickness was measured directly under a 10× microscope. Cellular infiltration was scored as follows: 0, no cells; 1, a few cells; 2, a ring of cells and 1 cell deep; 3, a ring of cells and 2–4 cells deep; and 4, a ring of cells and >4 cells deep. The scores of ten rats were averaged. Histopathology was assessed independently by three blinded pathologists.

Immunohistochemistry

To study mucus secretion in rat lungs in each group, an immunohistochemistry test was performed with the mucin antibody MUC5AC 21. Briefly, paraffin-embedded slides were deparaffinized with 100% xylene and rehydrated (100, 95, 90, 85, 80% ethanol). Subsequently, the deparaffinized slides were rehydrated and soaked in sodium citrate buffer to expose the antigen, followed by treatment with 3% hydrogen peroxide (10 min) to block endogenous peroxidase. After being washed with PBS, the slides were incubated with the MUC5AC antibody (Abcam, Cambridge, UK) in a 1:500 dilution of the original antibody dilution buffer (Beyotime, Shanghai, China ) for 12 h at 4℃. Secondary antibodies were detected using SignalStain DAB Substrate Kit (Cell Signaling Technology, Beverly, MA, USA). Slides were counterstained with hematoxylin (Ligen, Beijing, China) and mounted. Each group was examined using the positive fluorescence microscope DM6B (Leica, Frankfurt, Germany). Semi-quantitative image analysis was used to measure average optical density (AOD) to assess MUC5AC expression with Image Pro plus software version 6.0 (Media Cybernetics Corporation, USA).

Western blot

Western blot analysis was performed as previously described19. Briefly, lung tissue was homogenized using lysis buffer with 1 nM phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China), followed by centrifugation (8000g, 10 min) to collect the supernatant. The protein concentration in the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). For separated proteins, 300 μg of protein from each sample was loaded onto a gel for 10% SDS-PAGE. Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% bovine serum albumin (BSA) solution for 1 h, followed by overnight incubation with antibodies. primers anti-JAK2 (EPR23073-1, 1:1000, Abcam, Cambridge, UK), anti-phospho-JAK2 (EPR23028, 1:1000, Abcam, Cambridge, UK), anti-STAT3 (ab138483, 1 :1000, Abcam, Cambridge, UK) and anti-phospho-STAT3 (ab137521, 1:1000, Abcam, Cambridge, UK) at 4°C. Then, the membranes were continuously incubated with secondary antibodies (1:10,000 dilution in TBST containing 5% skimmed milk) and the bands were detected using a Western blot detection kit (Beyotime, Shanghai, China). The blot was scanned and analyzed using open source software ImageJ (downloaded free from https://imagej.nih.gov/ij/) to determine the band densitometry.

statistical analyzes

All data are presented as mean ± SD, and analyzes were performed with Graphpad Prism software version 8.0 for Win (GraphPad Software, San Diego, CA, USA). Comparisons between experimental groups were made using Tukey after one-way ANOVA for group-to-group comparison, p values ​​

Ethical approval and consent to participate

Laboratory animals were handled according to a protocol approved by the Center for Laboratory Animal Care, the Jiangsu Experimental Animal Ethics Committee and the Jiangsu Provincial Health Commission of China (Z2019053).