Efficacy of Pentoxifylline in Smoking Combined with Lipopolysaccharide Atomization Exposure Induced Emphysema in Mice

Efficacy of Pentoxifylline in Smoking Combined with Lipopolysaccharide Atomization Exposure Induced Emphysema in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of Pentoxifylline in Smoking Combined with Lipopolysaccharide Atomization Exposure Induced Emphysema in Mice Zhenghao Hu, Yaling Yu, Tianfeng Peng, Ruijie Niu, Zhuanyun Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7161599/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective To explore the effect of pentoxifylline dose and administration mode on lung pathology and inflammation induced by atomization exposure of cigarettes combined with lipopolysaccharides. Metholds: Female C57BL/6 mice were exposed to smoke (CS) + lipopolysaccharide (LPS) and pseudosmoke (SCS) for 10 weeks, and from week 9, animals were randomized into separate interventions with nebulized pentoxifylline (different doses), theophylline, budesonide suspension for 2 weeks, and a co-solvent control group was established. Animals are euthanized on the weekend of the 10th week. The ELISA method detected TNF-α, IL-8 and IL-1β expression in alveolar lavage fluid (BALF). After homogenization, the expression of MMP-12 and HDAC2 activity were detected by ELISA method; H&E staining of lung tissue sections to measure alveolar mean intercept (Lm) and alveolar destruction index (ADI); Reye-Jimsa staining assay for the determination of cell classification and quantity in BALF. Results The inflammatory reaction of lung after chronic CS + LPS exposure is enhanced, which is manifested as TNF-α,IL-8,IL-1 β and MMP-12 increase(CS + LPS vs SCS: TNF-α 68.70 ± 1.75pg/ml vs 32.67 ± 0.90pg/ml, p < 0.05; IL-8 45.66 ± 1.72pg/ml vs 15.72 ± 1.84pg/ml, p < 0.05; IL-1β 36.81 ± 1.02pg/ml vs 11.58 ± 0.76pg/ml, p < 0.05; MMP-12 103.57 ± 1.87ng/ml vs 31.96 ± 1.84ng/ml, p < 0.05). Furthermore, the HDAC2 activity decreased(CS + LPS vs SCS:8.86 ± 0.29U/ml vs 20.44 ± 0.60U/ml, p < 0.05), Lm and ADI increased(CS + LPS vs SCS: Lm,45.58 ± 0.50um vs 24.14 ± 2.93um, p < 0.05; ADI,51.90 ± 1.90% vs 6.29 ± 0.20%, p < 0.05).Meanwhile, the total cell count in BALF augmented(CS + LPS vs SCS:156.20 ± 18.88 10^5/ml vs 28.20 ± 3.50 10^5/ml, p < 0.05). Budesonide suspension has no significant effect on HDAC2 activity. Different doses of pentoxifylline (PTX) and theophylline (THEO) can restore part of HDAC2 activity ( p < 0.05). Conclusions Aerosol inhalation of pentoxifylline and theophylline could reduce lung inflammation induced by cigarette smoke combined with lipopolysaccharide exposure, reduce the expression of TNF-α,IL-8,IL-1β and MMP-12,and restore the decrease in HDAC2 activity induced by long-term smoke and lipopolysaccharide exposure, while inhalation of budesonide suspension alone had no effect on the activity of lung HDAC2. The recovery of HDAC2 activity is related to the nebulized inhalation dose of pentoxifylline, but more experimental studies are needed to determine the optimal concentration. Pulmonary inflammation Cigarette smoke Pentoxifylline Histone deacetyltransferase 2 Cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory disease characterized by persistent airway symptoms and airflow limitation. It presents as slow, progressive airflow obstruction that is only partially reversible [ 1 ]. There are many triggers and risk factors for COPD, with smoking being the primary risk factor. Other environmental toxic particles, including dust, toxic fumes, vapors, and the burning of biomass fuels, are also significant contributors [ 2 , 3 ]. These risk factors lead to the development of the disease through various pathophysiological mechanisms within the body, such as oxidative stress, inflammation, extracellular matrix protein degradation, and alveolar cell death. COPD typically occurs in individuals with a long history of smoking, and its pathological changes generally include three conditions: emphysema, small airway inflammation and fibrosis, and mucus gland hyperplasia, with the most notable mucus gland hyperplasia occurring in the large airways [ 4 , 5 ]. Different lung pathologies in COPD present distinct clinical manifestations, mainly chronic bronchitis and emphysema. Chronic bronchitis is a chronic inflammatory disease of the large airways, while emphysema is a disease that weakens the distal airways and lung parenchyma. In this condition, the airspaces in the distal airways permanently expand, elastin fibers in the alveoli are lost, and alveolar tissue is irreversibly damaged, leading to a reduction in the area available for gas exchange and a decline in lung function. COPD is a complex disease with a long course, frequent exacerbations, and many associated comorbidities, such as cor pulmonale and pulmonary encephalopathy. Acute exacerbations of COPD and related complications significantly reduce the patient's quality of life and survival, severely impacting their prognosis [ 6 , 7 ]. Relevant literature indicates that long-term exposure to cigarette smoke and toxic particles in the environment exacerbates pulmonary inflammation, leading to the recruitment of various inflammatory cells (such as macrophages, neutrophils, and lymphocytes) to the lungs under the guidance of chemokines. This results in an increased release of inflammatory factors, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, matrix metalloproteinase-12 (MMP-12), and interleukin (IL)-8, ultimately leading to reduced blood supply to the affected areas, damage to lung parenchyma, destruction of the elastin fiber network, thinning of the alveolar walls, expansion, rupture, or formation of bullae in the alveolar cavities, and emphysema-like changes in the lung parenchyma [ 8 , 9 ]. In the prevention and treatment of COPD, smoking cessation is the most important measure [ 10 , 11 ]. However, smokers with emphysema continue to experience pulmonary inflammation for a long time after quitting smoking, and autoreactive T cells are present in the peripheral blood. The activation of T cells, along with pulmonary inflammation, contributes to the loss of lung function [ 12 , 13 ], leading to a gradual decline in the patient's quality of life. Studies have shown that COPD is the third leading cause of death globally. Due to the lack of effective drugs that can reverse the trend of lung function deterioration in COPD patients or improve long-term lung function decline, COPD poses a significant economic burden on healthcare systems [ 14 ]. The World Health Organization (WHO) predicts that the prevalence of COPD will continue to rise with the aging of certain populations worldwide. Corticosteroids can effectively improve symptoms in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but they do not significantly improve respiratory conditions in patients with stable COPD [ 15 ]. Budesonide (BUD) is a commonly used inhaled anti-inflammatory medication in clinical practice. It primarily exerts anti-inflammatory effects by activating its related receptors, recruiting histone deacetylase 2 (HDAC2), and inhibiting inflammatory signaling pathways such as nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) [ 16 ]. Studies have shown that the use of corticosteroids in patients with stable COPD does not effectively improve symptoms and lung function, which may be closely related to the reduced activity of HDAC2 in these patients [ 17 ]. Therefore, enhancing or restoring HDAC2 activity may be an effective way to improve the efficacy of corticosteroid treatment in COPD patients Theophylline (THEO) is widely used in clinical practice for the treatment of acute asthma and COPD exacerbations. Research shows that theophylline is a phosphodiesterase inhibitor (PDEI) that non-selectively inhibits various phosphodiesterases and exerts effects similar to β-receptor agonists. It causes bronchial smooth muscle relaxation, alleviates airway spasms, and helps relieve patients' dyspnea. However, theophylline has a narrow therapeutic window, and at therapeutic doses, it often causes side effects such as nausea, vomiting, arrhythmias, and hypotension [ 18 , 19 ]. Relevant studies indicate that lower concentrations of theophylline in serum help improve HDAC2 activity in the body, enhancing the sensitivity of corticosteroids and their anti-inflammatory effects [ 20 , 21 ]. Pentoxifylline (PTX) is another non-selective PDEI, primarily used in the treatment of cardiovascular diseases and peripheral vascular diseases (e.g., post-stroke sequelae, chronic embolic arteritis). Research has found that PTX has broad antioxidant and anti-inflammatory activities. It can significantly inhibit the release of TNF-α from alveolar macrophages induced by lipopolysaccharide (LPS), as well as suppress inflammation factors such as neutrophil chemotactic factors, MMP-9, and MMP-12 induced by TNF-α, thereby reducing LPS-induced lung inflammation in mice [ 22 , 23 ]. Recent studies have shown that intraperitoneal injection of PTX can reverse the decline in lung HDAC2 activity induced by chronic smoke exposure in mice, alleviating lung tissue inflammation [ 24 ]. Based on this, the present study hypothesizes that nebulized PTX may restore HDAC2 activity and possibly have a synergistic anti-inflammatory effect with corticosteroids. This experiment used a mouse model of emphysema induced by chronic cigarette smoke combined with lipopolysaccharide exposure. The study aimed to investigate the effects of nebulized pentoxifylline (at five different concentrations), theophylline, and budesonide suspension on the lung pathology and inflammation induced by cigarette smoke combined with lipopolysaccharide exposure. The goal is to explore the potential application of PTX nebulization in COPD patients Material and Methods Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (IACUC Number:3269). Inhalation system The inhalation chamber consists of a glass bottle and four sealed white plastic boxes, with each plastic box (exposure chamber) connected to the glass bottle by a similarly sized transfer pipe. One commercially available cigarette is burned in the glass bottle at a compressed air flow rate of 4.4 L/min for ventilation. Under these conditions, the cigarette burns for about 6 minutes. Lipopolysaccharide (LPS) is administered through a nebulizer for inhalation exposure. LPS, derived from Pseudomonas aeruginosa , is prepared as a 2.5 mg/ml stock solution and stored at -20°C. Before use, it is taken out, dissolved at 4°C, and 0.7 ml of the stock solution is diluted with PBS to a total volume of 20 ml. The 20 ml diluted solution is divided into 4 doses (5 ml each), placed in the nebulizer cup (which is situated inside the exposure chamber). The nebulizer is activated, and LPS is aerosolized for inhalation by the mice. Under these conditions, 5 ml of the aerosolized solution takes about 15 minutes to be nebulized. Mice in the exposure chamber are simultaneously exposed (systemically) to both cigarette smoke and aerosolized LPS from the burning room and nebulizer output(Fig. 1 ). Study design Ninety 6–8 week old C57BL/6 mice obtained from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China, were fed in a suitable environment for one week before being randomly assigned to groups for a 10-week chronic cigarette smoke combined with lipopolysaccharide (CS + LPS) exposure (n = 80) or sham smoke/air (SCS) exposure (n = 10). At the end of week 8 of the experiment, all mice exposed to cigarette smoke and lipopolysaccharide were randomly assigned to receive drug interventions. The mice were divided into 9 groups (n = 10 for each group): the SCS group (blank group), the CS + LPS group (control group), the CS + LPS + PTX group (pentoxifylline intervention group, with concentrations of PTX1 to PTX5 at 9.8 mg/ml, 14.7 mg/ml, 22.0 mg/ml, 33.0 mg/ml, and 49.5 mg/ml, respectively), the CS + LPS + THEO group (theophylline intervention group), and the CS + LPS + BUD group (budesonide intervention group). Exposure protocol Exposure protocol The animals received a balanced diet and water ad libitum throughout the study. The mice in the experimental groups were exposed to cigarette smoke and lipopolysaccharide for approximately 1 hour each day, receiving 10 cigarettes for burning and 20 ml of lipopolysaccharide solution for nebulization. The mice allocated to the sham CS exposure were caged in the same environment but exposed only to fresh air for 60 min. These 60-min CS + LPS and SCS exposure episodes were conducted once a day, 5 days a week, for 10 weeks. Interventional medication Drug Administration Route: All drugs were administered via nebulization. Drug Dosage: The dosages of pentoxifylline, theophylline, and budesonide suspension were calculated based on dose conversion formulas for humans and animals from related literature [ 25 , 26 ]. Specifically, PTX (divided into 5 groups: PTX1-PTX5, concentrations: 9.8 mg/ml, 14.7 mg/ml, 22.0 mg/ml, 33.0 mg/ml, 49.5 mg/ml) was dissolved in sterile PBS to form a suspension for nebulization; theophylline (25 mg/ml) was also dissolved in sterile PBS to form a suspension for nebulization; inhaled budesonide suspension (0.5 mg/ml) was nebulized. Intervention Time: The interventions with pentoxifylline, theophylline, and inhaled budesonide suspension were conducted during the 9th and 10th weeks of CS + LPS exposure, lasting for two weeks. Treatment was administered 5 days a week, with daily dosing 1 hour before CS + LPS exposure. Meanwhile, the blank group (SCS) and the control group (CS + LPS) received nebulization with a solvent (5 ml sterile PBS) for the same duration as the treatment groups. Morphological studies At the end of week 10, 24 hours after the final CS + LPS exposure, mice were euthanized according to the updated American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. This was achieved via intraperitoneal injection of pentobarbital sodium (100 mg/kg body weight), followed by exsanguination via severing the abdominal aorta. The histological procedures were performed following the way reported previously [ 27 ]. The lungs were fixed for 48 h by intratracheal instillation of buffered formalin solution at a transpulmonary pressure of 20 cmH2O. Sagittal sections were taken from the left lung and processed by routine histological procedures for paraffin embedding. Five-µm thick histological sections were stained with hematoxylin-eosin and the combination of Schiff’s periodic acid and Alcian blue, pH 2.5. According to the method described by Zheng Xiaofang et al. [ 27 ], the morphological parameters of the lung tissue of all experimental mice were measured using a Stepanizer, which include the Alveolar Destruction Index (ADI) and Mean Linear Intercept (Lm). Pathological alterations and alveolar destruction were identified by the determination of the mean linear intercept (Lm) and alveolar destruction index (ADI). BALF and biomarker assessment The adjacent lungs underwent intratracheal instillation of 0.5 ml of ice-cold phosphate-buffered saline (PBS), three times, while the contralateral main bronchus was ligated. The bronchoalveolar lavage fluid (BALF) was recovered by gentle aspiration and subjected to filtration and centrifugation at 750 g for 10 min at 4℃. The supernatant was stored at − 80℃ for the subsequent measurements. The expression of IL-8, TNF-α and IL-1βin the BALF was measured using a commercial ELISA kit (Neobioscience) according to the manufacturer's instructions. The cell pellet was resuspended in 1.0 ml of sterile PBS (1×), and 10 µl of the resuspended solution was taken for cell counting under a microscope using a hemocytometer. The remaining resuspended solution was centrifuged again at 1500 rpm, 4°C, for about 10 minutes. After centrifugation, the supernatant was removed, but not completely, and based on the results of the cell count from the first step and the number of slides required, the remaining supernatant was used to resuspend the cell pellet. The resuspended cell pellet was smeared onto a glass slide, air-dried naturally, and then fixed in 10% neutral formalin solution for about 10 minutes. Following fixation, the smear was subjected to routine Wright-Giemsa staining. After staining, the slide was quickly dried in a 60°C oven, then placed in xylene for 5 minutes, followed by mounting with neutral balsam. The slide was then examined under an optical microscope, first under low magnification and then with an oil immersion lens. The lung tissue that underwent alveolar lavage was cut and stored at -80°C for later analysis. After retrieving the frozen lung tissue from − 80°C, the animal tissue weight was measured accurately. A 1:9 weight (mg) to volume (µl) ratio was used, and 0.9% sterile sodium chloride solution was added to the container. The tissue was then homogenized in an ice-water bath at 0–4°C using mechanical homogenization. After homogenization, the resulting homogenate (10%) was centrifuged at 2500–3000 rpm for 10 minutes. The supernatant was separated, and the pellet was discarded. The supernatant was stored for the subsequent measurements. The expression of MMP-12 and HDAC2 in the lung tissue was measured using a commercial ELISA kit (Neobioscience) according to the manufacturer's instructions. Statistical analysis Data were analyzed statistically using SPSS 22.0 and Prism 8.0. The normal distribution of the data was assessed using the D'Agostino and Pearson omnibus K2 normality test along with tests for homogeneity of variance. When the data met the assumptions of normality and homogeneity of variance, parametric tests (such as one-way ANOVA and t-tests) were used. When the data did not meet the assumptions of normality and homogeneity of variance, non-parametric tests (such as Kruskal-Wallis test and Mann-Whitney test) were applied. Bar graphs comparing multiple groups were created using one-way ANOVA in Prism 8.0. A p-value of < 0.05 was considered statistically significant. RESULTS Pulmonary morphology and morphometry As expected, the experimental mice exposed to both lipopolysaccharide (LPS) and cigarette smoke showed a significant increase in the mean linear intercept (Lm) (p < 0.05) (Fig. 1 , panel A and B, Table 1 ), and the alveolar destruction index (ADI) also significantly increased (p < 0.05) (Fig. 1 , panel C and D, Table 1 ). Compared to the control (CS + LPS) group, the drug intervention groups showed no significant change in the mean linear intercept after 2 weeks of treatment. However, the alveolar destruction index decreased to varying degrees (p 0.05) (Fig. 1 , panel C and D). Table 1 Measurements of biological variables in the control and study groups (data presented as mean ± SD) Group(n = 10) Lm(um) ADI(%) Total cells(10^5/ml) Neutrophils(10^5/ml) Mcrophages(10^5/ml) Lymphocytes(10^5/ml) Eosinophils(10^5/ml) SCS 24.14 ± 2.93 6.29 ± 0.20 28.20 ± 3.50 2.25 ± 0.28 23.68 ± 2.94 1.18 ± 0.15 0.26 ± 0.03 CS + LPS 45.58 ± 0.50 # 51.90 ± 1.90 # 156.20 ± 18.88 # 12.45 ± 1.51 # 131.1 ± 15.86 # 6.53 ± 0.15 # 1.42 ± 0.17 # CS + LPS + PTX5 43.40 ± 1.31 34.81 ± 1.05 * 77.25 ± 4.47 * 6.61 ± 0.36 * 64.87 ± 3.76 * 3.23 ± 0.19 * 0.70 ± 0.04 * CS + LPS + PTX4 43.16 ± 1.51 34.78 ± 1.00 * 79.79 ± 3.17 * 6.36 ± 0.25 * 67.01 ± 2.66 * 3.34 ± 0.13 * 0.73 ± 0.03 * CS + LPS + PTX3 43.04 ± 1.42 34.43 ± 0.81 * 68.08 ± 9.89 * 5.43 ± 0.79 * 57.18 ± 8.31 * 2.85 ± 0.41 * 0.62 ± 0.09 * CS + LPS + PTX2 44.05 ± 1.27 35.08 ± 0.30 * 73.08 ± 3.66 * 5.83 ± 0.29 * 61.37 ± 3.07 * 3.06 ± 0.15 * 0.66 ± 0.03 * CS + LPS + PTX1 44.29 ± 1.22 35.61 ± 0.63 * 79.71 ± 7.52 * 6.36 ± 0.60 * 66.94 ± 6.31 * 3.33 ± 0.31 * 0.72 ± 0.07* CS + LPS + THEO 44.32 ± 1.20 35.12 ± 1.11 * 76.16 ± 4.64 * 6.07 ± 0.37 * 63.95 ± 3.90 * 3.19 ± 0.19 * 0.69 ± 0.04 * CS + LPS + BUD 45.23 ± 1.17 36.55 ± 0.79 * 65.72 ± 4.46 * 5.24 ± 0.28 * 55.19 ± 2.00 * 2.75 ± 0.14 * 0.60 ± 0.03 * Abbreviations: SCS, shame cigarette smoke; CS, cigarette smoke; LPS, Lipopolysaccharide; PTX, pentoxifylline; THEO, theophylline; BUD, Budesonide; n = 10 for each group; Lm, mean linear intercept; ADI, Alveolar destruction index. # Compared to the counterpart variable in SCS exposure control group, p < 0.05. * Compared to the counterpart variable in CS + LPS exposure control group, p < 0.05. Pro- inflammatory cytokines in BALF The 10-week CS + LPS exposure induced the up-regulation of TNF- α, IL- 8 and IL-1β in BALF. The expression of IL-8 and IL-1β could be down-regulated by PTX, THEO or BUD monotherapy, whereas the expression of TNF-α was only decreased using PTX and BUD (p < 0.05) (Fig. 2 , Table 2 ). Treatment with different concentrations of PTX significantly reduced the expression of the aforementioned inflammatory factors in the BALF. The effect was most pronounced at a PTX concentration of 22.0 mg/ml, although the differences between the treatment concentration groups were small (p>0.05). Table 2 Measurements of biological variables in the control and study groups (data presented as mean ± SD) Group(n = 10) TNF-α(pg/ml) IL-8(pg/ml) IL-1β(pg/ml) MMP-12(ng/ml) HDAC2(U/ml) SCS 32.67 ± 0.90 15.72 ± 1.84 11.58 ± 0.76 31.96 ± 1.84 20.44 ± 0.60 CS + LPS 68.70 ± 1.75 # 45.66 ± 1.72 # 36.81 ± 1.02 # 103.57 ± 1.87 # 8.86 ± 0.29 # CS + LPS + PTX5 48.71 ± 1.12 * 28.74 ± 1.42 * 27.63 ± 1.72 *$ 66.47 ± 0.96 * & 14.50 ± 0.28 *^ CS + LPS + PTX4 51.19 ± 1.33 * 27.54 ± 2.36 * 27.98 ± 1.30 *$ 68.61 ± 2.66 * & 15.68 ± 1.00 *^ CS + LPS + PTX3 46.68 ± 0.87 * 26.15 ± 1.53 * 26.61 ± 0.99 *$ 58.40 ± 10.43 *& 16.60 ± 1.16 * ^ CS + LPS + PTX2 50.31 ± 2.10 * 29.89 ± 2.54 * 28.60 ± 0.95 *$ 64.71 ± 1.36 * & 16.06 ± 0.34 *^ CS + LPS + PTX1 54.58 ± 7.76 * 32.17 ± 2.17 * 30.48 ± 0.94 *$ 72.15 ± 1.31 * & 14.07 ± 0.27 * ^ CS + LPS + THEO 56.24 ± 3.57 * 29.90 ± 1.73 * 27.42 ± 0.90 *$ 84.74 ± 2.38 * 16.22 ± 0.24 * ^ CS + LPS + BUD 53.54 ± 2.21 * 24.78 ± 1.54 * 21.67 ± 1.37 * 70.22 ± 1.06 *$ 10.54 ± 0.18 Abbreviations: SCS, shame cigarette smoke; CS, cigarette smoke; LPS, Lipopolysaccharide; PTX, pentoxifylline; THEO, theophylline; BUD, Budesonide; n = 10 for each group; TNF-α, Tumor necrosis factor alpha;IL-8, Interleukin-8༛IL-1β, Interleukin-1β༛MMP-12, Matrix metallopeptidase 12༛HDAC2, Histone Deacetylase-2. # Compared to the counterpart variable in SCS exposure control group, p < 0.05. * Compared to the counterpart variable in CS + LPS exposure control group, p < 0.05. $ Compared to the counterpart variable in BUD intervention group, p < 0.05. & Compared to the counterpart variable in THEO and PTX intervention group, p < 0.05. $ Compared to the counterpart variable in THEO and BUD intervention group, p < 0.05. ^ Compared to the counterpart variable in BUD intervention group, p < 0.05. MMP-12 and HDAC- 2 activity in lung tissue Similar to the inflammatory factors in bronchoalveolar lavage fluid, the concentration of MMP-12 in lung tissue was significantly increased in the CS + LPS group of mice (p < 0.05) (Fig. 3 , panel A, Table 2 ). PTX, THEO, and BUD all downregulated the expression of MMP-12, with PTX showing a significantly stronger effect than THEO at different concentrations (p < 0.05) (Fig. 3 , panel A). Among the five concentrations of PTX tested, the downregulation of MMP-12 expression was most pronounced at a PTX concentration of 22.0 mg/ml, although the intragroup differences were minimal (p > 0.05). In contrast to the expression of MMP-12 in lung tissue, HDAC2 activity in the lung tissue of the CS + LPS group was significantly lower than that in the SCS group (p < 0.05) (Fig. 3 , panel B, Table 2 ). All intervention groups, except the BUD intervention group, significantly enhanced HDAC2 activity in the mouse lung tissue (p < 0.05) (Fig. 3 , panel B). Among these, the PTX intervention group at 22.0 mg/mL demonstrated a more pronounced upregulation of HDAC2 activity, although no significant differences were observed between the groups (p > 0.05). Cell Classification and Counting in BALF After 10 weeks of exposure to cigarette smoke and LPS, the total cell counts and the differential cell counts (including neutrophils, macrophages, lymphocytes, and eosinophils) in the BALF of the CS + LPS group mice were significantly increased (Fig. 4 , Table 1 ). PTX, THEO, and BUD all downregulated the expression of inflammatory cells in BALF and reduced the total cell count (p 0.05). Additionally, the reduction in inflammatory cell numbers in BALF was most notable at a PTX concentration of 22.0 mg/ml, but intragroup differences remained small (p > 0.05) (Fig. 5 ). Discussion COPD is a common, debilitating, and irreversible inflammatory lung disease closely associated with smoking. Similar to many other respiratory diseases (e.g., asthma, bronchiectasis), airway inflammation plays a crucial role in the development and progression of COPD. Chronic airway inflammation is a key driver in this process, leading to an increased presence of innate and adaptive immune cells within the airway lumen, inducing small airway fibrosis, and causing airway narrowing and eventual obstruction, accompanied by structural changes throughout the respiratory tract [ 28 , 29 ]. This chronic airway inflammation results from a combination of internal and environmental factors. Internal factors primarily refer to genetic susceptibility (i.e., genetic predisposition influences individual sensitivity to external factors, with specific gene polymorphisms being associated with human phenotypic responses to environmental exposure). Environmental factors include exposure to risk factors present in living and working environments, such as smoke, microorganisms, allergens like pollen and mites, air pollutants, and toxic gases [ 30 , 31 ]. Due to persistent nonspecific immune responses, COPD patients have increased susceptibility to infections, including respiratory viral diseases, which are among the primary triggers of AECOPD. The progression of COPD and the severity of its clinical symptoms are closely linked to chronic pulmonary inflammation. AECOPD represents an exacerbation of the inflammatory process, characterized by heightened systemic inflammatory responses, leading to disease progression, complications, and a significantly increased risk of mortality [ 28 , 32 ]. Therefore, effective anti-inflammatory and anti-infective treatments are essential strategies for improving the course of COPD, reducing adverse health events, and lowering the risk of mortality. It is widely acknowledged that the development and progression of COPD, as well as the severity of its clinical symptoms, are closely related to pulmonary inflammation and oxidative stress. The inflammatory response driven by an increased number of inflammatory cells is a key feature of COPD. Notably, this inflammatory response persists not only during acute exacerbations but also in stable phases of COPD [ 32 , 37 ]. Research has shown that 82% of acute exacerbations in COPD patients are caused by lower respiratory tract infections [ 38 ]. Bacterial infections exacerbate the recruitment of inflammatory cells to the lungs and increase oxidative stress, resulting in the production and release of pro-inflammatory cytokines such as reactive oxygen species (ROS), TNF-α, IL-8, IL-1, and matrix metalloproteinases (MMPs) [ 39 – 41 ]. TNF-α, as a regulatory factor of inflammation, works in conjunction with chemokines to trigger an inflammatory cascade, generating more inflammatory mediators while promoting the chemotaxis of inflammatory cells within the lungs. MMP-12, in particular, sustains and amplifies the inflammatory response, further exacerbating lung inflammation [ 39 , 42 ]. In many studies related to emphysema, "intervention treatments" often begin at the initial exposure of experimental animals to risk factors, before the emphysema model has developed. This approach differs from the clinical context of COPD patients, where treatment is administered after diagnosis. The former focuses on prevention, while the latter aims at treatment. Consequently, the results of such animal studies have limited relevance for guiding therapeutic strategies in patients already diagnosed with COPD [ 27 ]. Research has shown that intervention after the establishment of an animal disease model ("late-stage intervention") more accurately reflects the progression and treatment process of human diseases, offering insights into clinically relevant therapeutic strategies [ 36 ]. Previous studies using the "late-stage intervention" approach administered therapeutic drugs after cigarette smoke-induced emphysema had developed in mice and evaluated the "treatment" outcomes [ 24 ]. These studies demonstrated that "late-stage intervention" significantly alleviated pulmonary inflammation and oxidative stress, improved the general condition of the mice, and provided valuable insights for clinical COPD management. In this experiment, we also adopted a "late-stage intervention" strategy. After establishing the disease model (emphysema) in experimental animals, we conducted a two-week drug intervention under the induction conditions to investigate the therapeutic effects of PTX, THEO, and BUD on experimental emphysema in mice. Theophylline, a classic non-selective phosphodiesterase inhibitor (PDEI), is widely used in clinical settings for the treatment of acute asthma exacerbations. Studies have reported that theophylline at lower plasma concentrations can exhibit potential anti-inflammatory properties independent of its phosphodiesterase inhibition or antagonism of ADA receptors [ 45 ]. The regulation of inflammation via its effects on cAMP levels may be a key mechanism underlying its anti-inflammatory effects. In this study, theophylline was administered at a dose of 25 mg/ml. Compared to the control group, nebulized theophylline reduced the expression of IL-8, TNF-α, and IL-1β in BALF and decreased the total cell count in BALF. Similarly, Masato et al. [ 46 ] pretreated Hartley guinea pigs sensitized with ovalbumin (OVA) with aminophylline (25 mg/ml, nebulized) and investigated the airway and inflammatory responses upon subsequent OVA exposure. Their results showed that a single dose of nebulized aminophylline could not suppress eosinophil infiltration into the airways. However, repeated nebulization (twice daily for seven days) inhibited eosinophil infiltration and reduced the total cell count and eosinophil proportion in BALF. In the present study, nebulized theophylline at 25 mg/ml in a cigarette smoke and lipopolysaccharide-induced emphysema model resulted in a decrease in BALF total cell count, consistent with the findings of Masato et al. Additionally, nebulized theophylline significantly increased HDAC2 activity. Zheng et al. [ 24 ] demonstrated that intraperitoneal theophylline restored HDAC2 activity partially inhibited by long-term smoke exposure, significantly reduced ROS production, and showed a correlation between HDAC2 activity restoration and alleviation of oxidative stress. Thus, it is reasonable to believe that nebulized theophylline exerts anti-inflammatory effects and alleviates oxidative stress by restoring HDAC2 activity. PTX is also a non-selective phosphodiesterase inhibitor (PDEI) that effectively inhibits PDE3, PDE4, and PDE7 [ 47 ]. Studies have shown that PTX can specifically antagonize TNF-α, dose-dependently inhibiting the production of TNF-α in alveolar macrophages, as well as suppressing cytokine-induced neutrophil chemotactic factors, NF-κB, gelatinase B, and peroxidase. In inflammation, PTX exerts anti-inflammatory effects by downregulating the production of pro-inflammatory cytokines such as IL-6, IL-1, and IL-4 [ 48 – 50 ]. Additionally, PTX has broad antioxidant properties, and research indicates that it can alleviate oxidative stress induced by lipopolysaccharides, cigarette smoke, ozone, nitrogen mustard, and other agents [ 51 , 52 ]. In this study, after nebulized intervention with different concentrations of PTX, the expression of TNF-α, IL-8, and IL-1β in the BALF of mice was significantly reduced. PTX also notably enhanced HDAC2 activity and decreased MMP-12 expression in lung tissues. These findings align with those of Zheng et al. [ 24 ], despite the use of nebulized PTX in this study. To evaluate the effects of nebulized PTX on lung inflammation in smoke-exposed mice, five concentrations of PTX were tested. Although concentrations from PTX1 to PTX5 showed incremental increases, no linear relationship was observed between the concentrations and the monitored indicators. After comparing the levels of inflammatory factors in BALF, MMP-12 concentrations in lung tissues, HDAC2 activity in lung tissues, and morphological parameters, the third group (with a PTX concentration of 22.0 mg/ml) exhibited the most favorable therapeutic effects. Although the differences within the group were not statistically significant, this concentration showed better therapeutic effects compared to the others, suggesting that further animal studies are needed to determine the optimal nebulized concentration of PTX. In this study, PTX, THEO, and BUD all exhibited varying degrees of inhibition on the elevated levels of TNF-α, IL-8, IL-1β in the BALF and MMP-12 in lung tissue induced by chronic cigarette smoke and lipopolysaccharide exposure. Both PTX and THEO are phosphodiesterase inhibitors, and their effects on inflammatory factors and inflammatory cells were similar (with the exception of MMP-12, where PTX significantly downregulated its expression more than THEO). Both also had varying degrees of restoring HDAC2 activity in lung tissues. BUD, a corticosteroid commonly used in clinical anti-inflammatory treatments for conditions like AECOPD, asthma, and ARDS, significantly reduced the expression of TNF-α, IL-8, and IL-1β in BALF and the expression of MMP-12 in lung tissue. However, it did not enhance HDAC2 activity. This suggests that nebulized BUD alone does not restore HDAC2 activity. However, as mentioned in the introduction, BUD’s anti-inflammatory effects, mediated by inhibition of relevant signaling pathways, require the participation of HDAC2. Additionally, BUD is less effective in patients with stable COPD who have already experienced reduced HDAC2 activity. Since nebulized PTX and THEO significantly restore partial HDAC2 activity in lung tissues, a combination of BUD with PTX or THEO may provide better therapeutic outcomes and greater clinical benefits. However, further experiments are needed to validate this hypothesis. Conclusion In summary, nebulized PTX can alleviate lung inflammation and potentially reduce oxidative stress induced by chronic cigarette smoke and lipopolysaccharide exposure, while also restoring HDAC2 activity. THEO is also able to partially restore HDAC2 activity, whereas BUD does not restore HDAC2 activity. Furthermore, within the PTX intervention group, the CS + LPS + PTX3 group (22.0 mg/ml) exhibited a trend of superior performance in reducing inflammatory factor levels in BALF, decreasing MMP-12 concentrations in lung tissue, restoring HDAC2 activity, and improving lung tissue morphology compared to other concentrations of PTX within the group, although intragroup differences were minimal (p > 0.05). However, this study did not include roflumilast in the nebulization experiment, and due to time constraints, the combination of PTX and THEO with BUD for nebulization was not conducted. Additionally, exploring the appropriate nebulized concentration of PTX requires setting more concentration points to expand the concentration gradient. Therefore, future work should incorporate roflumilast, THEO, BUD, and PTX together in the study to observe the effects of individual nebulization versus combined nebulization with steroids. This will provide better experimental data and methodological insights for the clinical application of PTX inhalers. In conclusion, the results of this study suggest that nebulized PTX could potentially become a novel therapeutic agent for chronic inflammation in COPD, but further research is needed to determine the optimal dosage, administration methods, and the specific mechanisms of action. Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (Ethics Approval Number: [2021] IACUC No. 3269). The study strictly complied with the ARRIVE Guidelines 2.0 and relevant Chinese regulations for the ethical use of laboratory animals. All procedures were designed to minimize animal suffering, including the use of appropriate anesthesia, analgesia, and humane endpoints. The C57BL/6 mice were commercially obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). As animals were sourced from a professional laboratory animal provider and not privately owned, informed consent from owners was not required. Consent to participate is not applicable as this study utilized animal subjects. Consent for publication Not applicable. This manuscript does not contain any individual person's data. All experimental data derived from animal studies are published with the approval of the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (Approval ID: [2021] IACUC No. 3269). Availability of data and materials The data underlying this article cannot be shared publicly to protect the intellectual property rights of the study funder (CSPC Pharmaceutical Group Limited) under existing contractual agreements. De-identified data will be made available to qualified researchers upon reasonable request to the corresponding author ( [email protected] ) after approval by both Huazhong University of Science and Technology 's Data Access Committee and CSPC Pharmaceutical Group Limited. Specifications of the provided paclitaxel (Pentoxifylline) are available directly from CSPC Pharmaceutical Group Limited via [email protected] . Competing interests The authors report no conflict of interest. Funding This work was financially supported by CSPC Pharmaceutical Group Limited. The investigational drug paclitaxel (Pentoxifylline) was provided as an in-kind contribution by the same entity. Clinical trial number not applicable. Authors' contributions Hu: Conceptualization, Methodology, Investigation, Formal analysis, Writing Original Draft. Yu: Investigation, Data curation, Validation, Visualization. Peng: Resources, Software, Formal analysis. Niu: Validation, Investigation, Data curation. Li: Methodology, Resources. Wang: Supervision, Project administration. Zheng: Supervision, Project administration. Zhang*: Conceptualization, Supervision, Funding acquisition, Writing-Review & Editing, Project administration. All authors read and approved the final manuscript. Corresponding author Zhang is responsible for ensuring the accuracy and integrity of all research aspects and serves as the primary contact for communication. Acknowledgements The authors gratefully acknowledge the financial support provided by CSPC Pharmaceutical Group Limited for this research. References SAAD M I MCLEODL, HODGES C, et al. ADAM17 Deficiency Protects against Pulmonary Emphysema [J]. Am J Respir Cell Mol Biol. 2021;64(2):183–95. 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Am J Physiol Lung Cell Mol Physiol. 2018;315(3):L432–42. BARNES PJ, ADCOCK I M ITOK. Histone acetylation and deacetylation: importance in inflammatory lung diseases [J]. Eur Respir J. 2005;25(3):552–63. KAMATA H, HONDA S-I MAEDAS, et al. Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases [J]. Cell. 2005;120(5):649–61. BARNES PJ. Theophylline [J]. Am J Respir Crit Care Med. 2013;188(8):901–6. MURAKI M, WADA S, OHNO T, et al. Effects of inhaled aminophylline on airway constriction and inflammation in ovalbumin-sensitized guinea pigs [J]. Drug Deliv. 2014;21(5):321–7. ESSAYAN D M. Cyclic nucleotide phosphodiesterase (PDE) inhibitors and immunomodulation [J]. Biochem Pharmacol. 1999;57(9):965–73. DE CAMPOS T, DEREE J, MARTINS J O, et al. Pentoxifylline attenuates pulmonary inflammation and neutrophil activation in experimental acute pancreatitis [J]. Pancreas. 2008;37(1):42–9. DEREE J, MARTINS J, DE CAMPOS T, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7161599","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502694732,"identity":"4e0885fa-751b-41f3-82d1-6caa23d2315e","order_by":0,"name":"Zhenghao Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACNmb24x8+VLDJMfY3HyBOCx87TxrjjDN8xswzjiUQp0WOn8GMmbdNLrG9IceAWIcxpD3gbTNL7G048/HGGwY7Od0GgloYjxtInEszntncu9lyDkOysdkBwrYkSBiUHZPd2HB2mzQPw4HEbURoMZBIYPvPuP9AzjOitZhJHGhjU2xsyGEjVgtPsmHDGTZjxhnHjC3nGBDhF/n+4wcf/4FE5cMbbyrs5AhqQQESPERGDbIWUnWMglEwCkbBiAAAHRFBMBt9awEAAAAASUVORK5CYII=","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhenghao","middleName":"","lastName":"Hu","suffix":""},{"id":502694733,"identity":"6692d009-3359-4161-990e-9ca7790bd1de","order_by":1,"name":"Yaling Yu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yaling","middleName":"","lastName":"Yu","suffix":""},{"id":502694734,"identity":"7f34820b-3142-4a25-bb4f-6d6e448cbe64","order_by":2,"name":"Tianfeng Peng","email":"","orcid":"","institution":"Second Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tianfeng","middleName":"","lastName":"Peng","suffix":""},{"id":502694735,"identity":"868444ad-bb0b-4bc4-b8e7-8bbefd793684","order_by":3,"name":"Ruijie Niu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ruijie","middleName":"","lastName":"Niu","suffix":""},{"id":502694736,"identity":"6c296ea7-ee86-485d-b8ae-731862fddcaf","order_by":4,"name":"Zhuanyun Li","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhuanyun","middleName":"","lastName":"Li","suffix":""},{"id":502694737,"identity":"4dc09641-dc3c-4a82-a4ae-ac1f19974868","order_by":5,"name":"Wenjing Wang","email":"","orcid":"","institution":"Henan Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Wang","suffix":""},{"id":502694738,"identity":"fc2afa21-4036-4497-9f63-23008e4e89ff","order_by":6,"name":"Xiaofang Zheng","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaofang","middleName":"","lastName":"Zheng","suffix":""},{"id":502694740,"identity":"f9612f4e-a75c-483e-887f-02644b4850ed","order_by":7,"name":"Jinnong Zhang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jinnong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-19 03:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7161599/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7161599/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89660565,"identity":"bbd4a121-9fe7-415b-a65b-abe62879198d","added_by":"auto","created_at":"2025-08-22 11:06:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1152639,"visible":true,"origin":"","legend":"\u003cp\u003epanel A. Comparison of lung tissue pathological morphology. Chronic exposure to lipopolysaccharide (LPS) combined with cigarette smoke resulted in increased abnormal pathological structures in mouse lung tissues, including thinning of alveolar walls, and a significant number of alveoli becoming enlarged, ruptured, or fused to form bullae, presenting emphysema-like changes under microscopy. After drug intervention, the microscopic morphological changes in lung tissues of mice in each experimental group showed no significant difference from those in the control group (CS + LPS) (Hematoxylin-eosin staining, ×100 magnification).\u003c/p\u003e\n\u003cp\u003epanel B. Chronic exposure to cigarette smoke combined with lipopolysaccharide (LPS) significantly increases the mean linear intercept (Lm) in experimental mice. Compared with mice in the CS + LPS group, pentoxifylline (PTX), theophylline (THEO), and budesonide (BUD) show no significant effect on the mean linear intercept.\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. SCS.\u003c/p\u003e\n\u003cp\u003epanel C. Comparison of mouse lung tissue morphology under high-power microscopy. (●: Alveolar fusion, disruption/disappearance of alveolar septa, abnormal dilation of alveolar spaces, and formation of pulmonary bullae. \u003cstrong\u003e↑\u003c/strong\u003e: Residual fragments of lung parenchyma in alveolar ducts. ↑:Thinning, discontinuity, or disruption of alveolar walls. Staining: Hematoxylin-eosin staining; grayscale image; magnification: ×400.)\u003c/p\u003e\n\u003cp\u003epanel D. Chronic exposure to cigarette smoke combined with lipopolysaccharide (LPS) significantly increases the alveolar destruction index (ADI) in experimental mice. Compared with mice in the CS + LPS group, pentoxifylline (PTX), theophylline (THEO), and budesonide (BUD) all reduce the alveolar destruction index.\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. SCS;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. CS+LPS.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7161599/v1/f7c50326ca110ee8712a7b06.jpg"},{"id":89658546,"identity":"7491e12b-d0b1-4017-89c7-49a392b62c2a","added_by":"auto","created_at":"2025-08-22 10:42:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96203,"visible":true,"origin":"","legend":"\u003cp\u003eChronic exposure to cigarette smoke combined with lipopolysaccharide (LPS) results in a significant increase in the levels of proinflammatory cytokines TNF-α, IL-8, and IL-1β in bronchoalveolar lavage fluid (BALF). Both pentoxifylline (PTX) and budesonide (BUD) at different concentrations can down-regulate the expression of TNF-α; among them, the intervention group with PTX at a concentration of 22.0 mg/ml shows a more significant reduction in TNF-α expression, while there is no statistically significant difference between other intervention groups. Theophylline (THEO) can decrease the expression of TNF-α in BALF, but the difference is not statistically significant. PTX, THEO, and BUD all reduce the expressions of IL-8 and IL-1β in BALF, with BUD exerting the most significant effect in lowering IL-1β expression in BALF.\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. SCS; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. CS+LPS; \u003csup\u003e$\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. BUD.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7161599/v1/cedee62ae53b5a59643d7f9f.jpg"},{"id":89658547,"identity":"74769a33-16f1-4f4e-8a39-c41db8929f16","added_by":"auto","created_at":"2025-08-22 10:42:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77894,"visible":true,"origin":"","legend":"\u003cp\u003eChronic exposure to cigarette smoke combined with lipopolysaccharide (LPS) leads to a significant increase in the concentration of matrix metalloproteinase-12 (MMP-12) in lung tissues. Theophylline (THEO), budesonide (BUD), and pentoxifylline (PTX) at different concentrations can all down-regulate the expression of MMP-12. Among them, the CS+LPS+PTX3 group (22.0 mg/ml) shows a relatively more significant reduction in MMP-12 expression; however, there is no statistically significant difference between this group and other intervention groups, nor between this group and the CS+LPS+BUD group.The activity of histone deacetylase 2 (HDAC2) in mouse lung tissues decreases significantly after chronic exposure to cigarette smoke combined with LPS. Budesonide (BUD) fails to significantly restore HDAC2 activity, while theophylline (THEO) and pentoxifylline (PTX) at different concentrations can all enhance HDAC2 activity. Specifically, PTX at a concentration of 22.0 mg/ml results in a more notable enhancement of HDAC2 activity, but no statistically significant differences are observed between this group and other intervention groups, or between this group and the CS+LPS+THEO group.\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. SCS; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. CS+LPS; \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 VS. THEO; ^\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. BUD; \u003csup\u003e$\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7161599/v1/47fa86b998a08374e2f87916.jpg"},{"id":89659914,"identity":"bf32a36e-f364-4ef1-87bb-7fa3fce5927e","added_by":"auto","created_at":"2025-08-22 10:58:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107240,"visible":true,"origin":"","legend":"\u003cp\u003eCell classification staining of bronchoalveolar lavage fluid (BALF). (\u003cstrong\u003e↑\u003c/strong\u003e：Eosinophils; \u003cstrong\u003e↑\u003c/strong\u003e：Macrophages; \u003cstrong\u003e↑\u003c/strong\u003e：Neutrophils; \u003cstrong\u003e↑\u003c/strong\u003e：Lymphocytes. Staining: Wright-Giemsa stain; Magnification: ×400.)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7161599/v1/d6e22d011bc6af4f638cb3ed.jpg"},{"id":89658549,"identity":"bccb1826-afec-494d-b937-32b39d46dd0c","added_by":"auto","created_at":"2025-08-22 10:42:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96428,"visible":true,"origin":"","legend":"\u003cp\u003eChronic exposure to cigarette smoke combined with lipopolysaccharide (LPS) leads to a significant increase in the total number of cells and the counts of various inflammatory cells (neutrophils, macrophages, lymphocytes, and eosinophils) in the bronchoalveolar lavage fluid (BALF) of mice. Pentoxifylline (PTX), theophylline (THEO), and budesonide (BUD) all reduce the total cell number and the counts of each type of inflammatory cell in BALF. Among them, BUD shows a relatively more significant effect in reducing inflammatory cell counts, though the difference is not statistically significant. In the PTX intervention groups, the CS+LPS+PTX3 group (22.0 mg/ml) exhibits a more obvious reduction in inflammatory cell counts; however, there is no statistically significant difference within the groups.\u003csup\u003e #\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. SCS; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. CS+LPS.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7161599/v1/82f186f9ad458ab61cf8bf8b.jpg"},{"id":89661822,"identity":"a271f88e-6ce6-4f4a-9c33-ce1314ca4d5a","added_by":"auto","created_at":"2025-08-22 11:14:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2314468,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7161599/v1/e2075322-c72b-4d01-866e-0d22873146b6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEfficacy of Pentoxifylline in Smoking Combined with Lipopolysaccharide Atomization Exposure Induced Emphysema in Mice\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory disease characterized by persistent airway symptoms and airflow limitation. It presents as slow, progressive airflow obstruction that is only partially reversible [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. There are many triggers and risk factors for COPD, with smoking being the primary risk factor. Other environmental toxic particles, including dust, toxic fumes, vapors, and the burning of biomass fuels, are also significant contributors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These risk factors lead to the development of the disease through various pathophysiological mechanisms within the body, such as oxidative stress, inflammation, extracellular matrix protein degradation, and alveolar cell death. COPD typically occurs in individuals with a long history of smoking, and its pathological changes generally include three conditions: emphysema, small airway inflammation and fibrosis, and mucus gland hyperplasia, with the most notable mucus gland hyperplasia occurring in the large airways [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Different lung pathologies in COPD present distinct clinical manifestations, mainly chronic bronchitis and emphysema. Chronic bronchitis is a chronic inflammatory disease of the large airways, while emphysema is a disease that weakens the distal airways and lung parenchyma. In this condition, the airspaces in the distal airways permanently expand, elastin fibers in the alveoli are lost, and alveolar tissue is irreversibly damaged, leading to a reduction in the area available for gas exchange and a decline in lung function. COPD is a complex disease with a long course, frequent exacerbations, and many associated comorbidities, such as cor pulmonale and pulmonary encephalopathy. Acute exacerbations of COPD and related complications significantly reduce the patient's quality of life and survival, severely impacting their prognosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Relevant literature indicates that long-term exposure to cigarette smoke and toxic particles in the environment exacerbates pulmonary inflammation, leading to the recruitment of various inflammatory cells (such as macrophages, neutrophils, and lymphocytes) to the lungs under the guidance of chemokines. This results in an increased release of inflammatory factors, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, matrix metalloproteinase-12 (MMP-12), and interleukin (IL)-8, ultimately leading to reduced blood supply to the affected areas, damage to lung parenchyma, destruction of the elastin fiber network, thinning of the alveolar walls, expansion, rupture, or formation of bullae in the alveolar cavities, and emphysema-like changes in the lung parenchyma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the prevention and treatment of COPD, smoking cessation is the most important measure [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, smokers with emphysema continue to experience pulmonary inflammation for a long time after quitting smoking, and autoreactive T cells are present in the peripheral blood. The activation of T cells, along with pulmonary inflammation, contributes to the loss of lung function [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], leading to a gradual decline in the patient's quality of life. Studies have shown that COPD is the third leading cause of death globally. Due to the lack of effective drugs that can reverse the trend of lung function deterioration in COPD patients or improve long-term lung function decline, COPD poses a significant economic burden on healthcare systems [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The World Health Organization (WHO) predicts that the prevalence of COPD will continue to rise with the aging of certain populations worldwide.\u003c/p\u003e\u003cp\u003eCorticosteroids can effectively improve symptoms in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but they do not significantly improve respiratory conditions in patients with stable COPD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBudesonide (BUD) is a commonly used inhaled anti-inflammatory medication in clinical practice. It primarily exerts anti-inflammatory effects by activating its related receptors, recruiting histone deacetylase 2 (HDAC2), and inhibiting inflammatory signaling pathways such as nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Studies have shown that the use of corticosteroids in patients with stable COPD does not effectively improve symptoms and lung function, which may be closely related to the reduced activity of HDAC2 in these patients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, enhancing or restoring HDAC2 activity may be an effective way to improve the efficacy of corticosteroid treatment in COPD patients\u003c/p\u003e\u003cp\u003eTheophylline (THEO) is widely used in clinical practice for the treatment of acute asthma and COPD exacerbations. Research shows that theophylline is a phosphodiesterase inhibitor (PDEI) that non-selectively inhibits various phosphodiesterases and exerts effects similar to β-receptor agonists. It causes bronchial smooth muscle relaxation, alleviates airway spasms, and helps relieve patients' dyspnea. However, theophylline has a narrow therapeutic window, and at therapeutic doses, it often causes side effects such as nausea, vomiting, arrhythmias, and hypotension [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Relevant studies indicate that lower concentrations of theophylline in serum help improve HDAC2 activity in the body, enhancing the sensitivity of corticosteroids and their anti-inflammatory effects [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Pentoxifylline (PTX) is another non-selective PDEI, primarily used in the treatment of cardiovascular diseases and peripheral vascular diseases (e.g., post-stroke sequelae, chronic embolic arteritis). Research has found that PTX has broad antioxidant and anti-inflammatory activities. It can significantly inhibit the release of TNF-α from alveolar macrophages induced by lipopolysaccharide (LPS), as well as suppress inflammation factors such as neutrophil chemotactic factors, MMP-9, and MMP-12 induced by TNF-α, thereby reducing LPS-induced lung inflammation in mice [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent studies have shown that intraperitoneal injection of PTX can reverse the decline in lung HDAC2 activity induced by chronic smoke exposure in mice, alleviating lung tissue inflammation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Based on this, the present study hypothesizes that nebulized PTX may restore HDAC2 activity and possibly have a synergistic anti-inflammatory effect with corticosteroids. This experiment used a mouse model of emphysema induced by chronic cigarette smoke combined with lipopolysaccharide exposure. The study aimed to investigate the effects of nebulized pentoxifylline (at five different concentrations), theophylline, and budesonide suspension on the lung pathology and inflammation induced by cigarette smoke combined with lipopolysaccharide exposure. The goal is to explore the potential application of PTX nebulization in COPD patients\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthics approval\u003c/h2\u003e\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (IACUC Number:3269).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInhalation system\u003c/h3\u003e\n\u003cp\u003eThe inhalation chamber consists of a glass bottle and four sealed white plastic boxes, with each plastic box (exposure chamber) connected to the glass bottle by a similarly sized transfer pipe. One commercially available cigarette is burned in the glass bottle at a compressed air flow rate of 4.4 L/min for ventilation. Under these conditions, the cigarette burns for about 6 minutes. Lipopolysaccharide (LPS) is administered through a nebulizer for inhalation exposure. LPS, derived from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, is prepared as a 2.5 mg/ml stock solution and stored at -20\u0026deg;C. Before use, it is taken out, dissolved at 4\u0026deg;C, and 0.7 ml of the stock solution is diluted with PBS to a total volume of 20 ml. The 20 ml diluted solution is divided into 4 doses (5 ml each), placed in the nebulizer cup (which is situated inside the exposure chamber). The nebulizer is activated, and LPS is aerosolized for inhalation by the mice. Under these conditions, 5 ml of the aerosolized solution takes about 15 minutes to be nebulized. Mice in the exposure chamber are simultaneously exposed (systemically) to both cigarette smoke and aerosolized LPS from the burning room and nebulizer output(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eNinety 6\u0026ndash;8 week old C57BL/6 mice obtained from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China, were fed in a suitable environment for one week before being randomly assigned to groups for a 10-week chronic cigarette smoke combined with lipopolysaccharide (CS\u0026thinsp;+\u0026thinsp;LPS) exposure (n\u0026thinsp;=\u0026thinsp;80) or sham smoke/air (SCS) exposure (n\u0026thinsp;=\u0026thinsp;10). At the end of week 8 of the experiment, all mice exposed to cigarette smoke and lipopolysaccharide were randomly assigned to receive drug interventions. The mice were divided into 9 groups (n\u0026thinsp;=\u0026thinsp;10 for each group): the SCS group (blank group), the CS\u0026thinsp;+\u0026thinsp;LPS group (control group), the CS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX group (pentoxifylline intervention group, with concentrations of PTX1 to PTX5 at 9.8 mg/ml, 14.7 mg/ml, 22.0 mg/ml, 33.0 mg/ml, and 49.5 mg/ml, respectively), the CS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;THEO group (theophylline intervention group), and the CS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;BUD group (budesonide intervention group).\u003c/p\u003e\n\u003ch3\u003eExposure protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eExposure protocol\u003c/div\u003e\u003cp\u003eThe animals received a balanced diet and water ad libitum throughout the study. The mice in the experimental groups were exposed to cigarette smoke and lipopolysaccharide for approximately 1 hour each day, receiving 10 cigarettes for burning and 20 ml of lipopolysaccharide solution for nebulization. The mice allocated to the sham CS exposure were caged in the same environment but exposed only to fresh air for 60 min. These 60-min CS\u0026thinsp;+\u0026thinsp;LPS and SCS exposure episodes were conducted once a day, 5 days a week, for 10 weeks.\u003c/p\u003e\n\u003ch3\u003eInterventional medication\u003c/h3\u003e\n\u003cp\u003eDrug Administration Route: All drugs were administered via nebulization.\u003c/p\u003e\u003cp\u003eDrug Dosage: The dosages of pentoxifylline, theophylline, and budesonide suspension were calculated based on dose conversion formulas for humans and animals from related literature [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Specifically, PTX (divided into 5 groups: PTX1-PTX5, concentrations: 9.8 mg/ml, 14.7 mg/ml, 22.0 mg/ml, 33.0 mg/ml, 49.5 mg/ml) was dissolved in sterile PBS to form a suspension for nebulization; theophylline (25 mg/ml) was also dissolved in sterile PBS to form a suspension for nebulization; inhaled budesonide suspension (0.5 mg/ml) was nebulized.\u003c/p\u003e\u003cp\u003eIntervention Time: The interventions with pentoxifylline, theophylline, and inhaled budesonide suspension were conducted during the 9th and 10th weeks of CS\u0026thinsp;+\u0026thinsp;LPS exposure, lasting for two weeks. Treatment was administered 5 days a week, with daily dosing 1 hour before CS\u0026thinsp;+\u0026thinsp;LPS exposure. Meanwhile, the blank group (SCS) and the control group (CS\u0026thinsp;+\u0026thinsp;LPS) received nebulization with a solvent (5 ml sterile PBS) for the same duration as the treatment groups.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMorphological studies\u003c/h2\u003e\u003cp\u003e At the end of week 10, 24 hours after the final CS\u0026thinsp;+\u0026thinsp;LPS exposure, mice were euthanized according to the updated American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. This was achieved via intraperitoneal injection of pentobarbital sodium (100 mg/kg body weight), followed by exsanguination via severing the abdominal aorta. The histological procedures were performed following the way reported previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The lungs were fixed for 48 h by intratracheal instillation of buffered formalin solution at a transpulmonary pressure of 20 cmH2O. Sagittal sections were taken from the left lung and processed by routine histological procedures for paraffin embedding. Five-\u0026micro;m thick histological sections were stained with hematoxylin-eosin and the combination of Schiff\u0026rsquo;s periodic acid and Alcian blue, pH 2.5. According to the method described by Zheng Xiaofang et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the morphological parameters of the lung tissue of all experimental mice were measured using a Stepanizer, which include the Alveolar Destruction Index (ADI) and Mean Linear Intercept (Lm). Pathological alterations and alveolar destruction were identified by the determination of the mean linear intercept (Lm) and alveolar destruction index (ADI).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBALF and biomarker assessment\u003c/h3\u003e\n\u003cp\u003eThe adjacent lungs underwent intratracheal instillation of 0.5 ml of ice-cold phosphate-buffered saline (PBS), three times, while the contralateral main bronchus was ligated. The bronchoalveolar lavage fluid (BALF) was recovered by gentle aspiration and subjected to filtration and centrifugation at 750 g for 10 min at 4℃. The supernatant was stored at \u0026minus;\u0026thinsp;80℃ for the subsequent measurements. The expression of IL-8, TNF-α and IL-1βin the BALF was measured using a commercial ELISA kit (Neobioscience) according to the manufacturer's instructions.\u003c/p\u003e\u003cp\u003eThe cell pellet was resuspended in 1.0 ml of sterile PBS (1\u0026times;), and 10 \u0026micro;l of the resuspended solution was taken for cell counting under a microscope using a hemocytometer. The remaining resuspended solution was centrifuged again at 1500 rpm, 4\u0026deg;C, for about 10 minutes. After centrifugation, the supernatant was removed, but not completely, and based on the results of the cell count from the first step and the number of slides required, the remaining supernatant was used to resuspend the cell pellet. The resuspended cell pellet was smeared onto a glass slide, air-dried naturally, and then fixed in 10% neutral formalin solution for about 10 minutes. Following fixation, the smear was subjected to routine Wright-Giemsa staining. After staining, the slide was quickly dried in a 60\u0026deg;C oven, then placed in xylene for 5 minutes, followed by mounting with neutral balsam. The slide was then examined under an optical microscope, first under low magnification and then with an oil immersion lens.\u003c/p\u003e\u003cp\u003eThe lung tissue that underwent alveolar lavage was cut and stored at -80\u0026deg;C for later analysis. After retrieving the frozen lung tissue from \u0026minus;\u0026thinsp;80\u0026deg;C, the animal tissue weight was measured accurately. A 1:9 weight (mg) to volume (\u0026micro;l) ratio was used, and 0.9% sterile sodium chloride solution was added to the container. The tissue was then homogenized in an ice-water bath at 0\u0026ndash;4\u0026deg;C using mechanical homogenization. After homogenization, the resulting homogenate (10%) was centrifuged at 2500\u0026ndash;3000 rpm for 10 minutes. The supernatant was separated, and the pellet was discarded. The supernatant was stored for the subsequent measurements. The expression of MMP-12 and HDAC2 in the lung tissue was measured using a commercial ELISA kit (Neobioscience) according to the manufacturer's instructions.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed statistically using SPSS 22.0 and Prism 8.0. The normal distribution of the data was assessed using the D'Agostino and Pearson omnibus K2 normality test along with tests for homogeneity of variance. When the data met the assumptions of normality and homogeneity of variance, parametric tests (such as one-way ANOVA and t-tests) were used. When the data did not meet the assumptions of normality and homogeneity of variance, non-parametric tests (such as Kruskal-Wallis test and Mann-Whitney test) were applied. Bar graphs comparing multiple groups were created using one-way ANOVA in Prism 8.0. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePulmonary morphology and morphometry\u003c/h2\u003e\u003cp\u003eAs expected, the experimental mice exposed to both lipopolysaccharide (LPS) and cigarette smoke showed a significant increase in the mean linear intercept (Lm) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel A and B, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the alveolar destruction index (ADI) also significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel C and D, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to the control (CS\u0026thinsp;+\u0026thinsp;LPS) group, the drug intervention groups showed no significant change in the mean linear intercept after 2 weeks of treatment. However, the alveolar destruction index decreased to varying degrees (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel A and B). All drug intervention groups were able to reduce the ADI, but the differences within and between the groups were small (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel C and D).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMeasurements of biological variables in the control and study groups (data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLm(um)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eADI(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTotal cells(10^5/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNeutrophils(10^5/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMcrophages(10^5/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLymphocytes(10^5/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eEosinophils(10^5/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e24.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e28.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e23.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e45.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e51.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e156.20\u0026thinsp;\u0026plusmn;\u0026thinsp;18.88\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e12.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e131.1\u0026thinsp;\u0026plusmn;\u0026thinsp;15.86\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e43.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e34.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e77.25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.47\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e64.87\u0026thinsp;\u0026plusmn;\u0026thinsp;3.76\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e43.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e34.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e79.79\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e67.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e43.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e34.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e68.08\u0026thinsp;\u0026plusmn;\u0026thinsp;9.89\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e57.18\u0026thinsp;\u0026plusmn;\u0026thinsp;8.31\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e44.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e73.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.66\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e61.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.07\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e44.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e79.71\u0026thinsp;\u0026plusmn;\u0026thinsp;7.52\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e66.94\u0026thinsp;\u0026plusmn;\u0026thinsp;6.31\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;THEO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e44.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e35.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e76.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.64\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e63.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;BUD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e45.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e36.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e65.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e55.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eAbbreviations: SCS, shame cigarette smoke; CS, cigarette smoke; LPS, Lipopolysaccharide; PTX, pentoxifylline; THEO, theophylline; BUD, Budesonide; n\u0026thinsp;=\u0026thinsp;10 for each group; Lm, mean linear intercept; ADI, Alveolar destruction index.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e#\u003c/sup\u003eCompared to the counterpart variable in SCS exposure control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e*\u003c/sup\u003eCompared to the counterpart variable in CS\u0026thinsp;+\u0026thinsp;LPS exposure control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePro- inflammatory cytokines in BALF\u003c/h2\u003e\u003cp\u003eThe 10-week CS\u0026thinsp;+\u0026thinsp;LPS exposure induced the up-regulation of TNF- α, IL- 8 and IL-1β in BALF. The expression of IL-8 and IL-1β could be down-regulated by PTX, THEO or BUD monotherapy, whereas the expression of TNF-α was only decreased using PTX and BUD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Treatment with different concentrations of PTX significantly reduced the expression of the aforementioned inflammatory factors in the BALF. The effect was most pronounced at a PTX concentration of 22.0 mg/ml, although the differences between the treatment concentration groups were small (p\u0026gt;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMeasurements of biological variables in the control and study groups (data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTNF-α(pg/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIL-8(pg/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIL-1β(pg/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMMP-12(ng/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHDAC2(U/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e31.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e20.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e68.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e45.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e36.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e103.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e8.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e48.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e28.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e27.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e66.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003e* \u0026amp;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e14.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003e*^\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e51.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e27.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e27.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e68.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003csup\u003e* \u0026amp;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e15.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003e*^\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e46.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e26.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e26.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e58.40\u0026thinsp;\u0026plusmn;\u0026thinsp;10.43\u003csup\u003e*\u0026amp;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e16.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003csup\u003e* ^\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e50.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e29.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e28.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e64.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003csup\u003e* \u0026amp;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e16.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003e*^\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e54.58\u0026thinsp;\u0026plusmn;\u0026thinsp;7.76\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e32.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e30.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e72.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003csup\u003e* \u0026amp;\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e14.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003e* ^\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;THEO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e56.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e29.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e27.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e84.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e16.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e* ^\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;BUD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e53.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e24.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e70.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003e*$\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e10.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eAbbreviations: SCS, shame cigarette smoke; CS, cigarette smoke; LPS, Lipopolysaccharide; PTX, pentoxifylline; THEO, theophylline; BUD, Budesonide; n\u0026thinsp;=\u0026thinsp;10 for each group; TNF-α, Tumor necrosis factor alpha;IL-8, Interleukin-8༛IL-1β, Interleukin-1β༛MMP-12, Matrix metallopeptidase 12༛HDAC2, Histone Deacetylase-2.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e#\u003c/sup\u003eCompared to the counterpart variable in SCS exposure control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e*\u003c/sup\u003eCompared to the counterpart variable in CS\u0026thinsp;+\u0026thinsp;LPS exposure control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e$\u003c/sup\u003eCompared to the counterpart variable in BUD intervention group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e\u0026amp;\u003c/sup\u003eCompared to the counterpart variable in THEO and PTX intervention group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e$\u003c/sup\u003eCompared to the counterpart variable in THEO and BUD intervention group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e^\u003c/sup\u003eCompared to the counterpart variable in BUD intervention group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMMP-12 and HDAC- 2 activity in lung tissue\u003c/h2\u003e\u003cp\u003eSimilar to the inflammatory factors in bronchoalveolar lavage fluid, the concentration of MMP-12 in lung tissue was significantly increased in the CS\u0026thinsp;+\u0026thinsp;LPS group of mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel A, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PTX, THEO, and BUD all downregulated the expression of MMP-12, with PTX showing a significantly stronger effect than THEO at different concentrations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel A). Among the five concentrations of PTX tested, the downregulation of MMP-12 expression was most pronounced at a PTX concentration of 22.0 mg/ml, although the intragroup differences were minimal (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast to the expression of MMP-12 in lung tissue, HDAC2 activity in the lung tissue of the CS\u0026thinsp;+\u0026thinsp;LPS group was significantly lower than that in the SCS group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel B, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All intervention groups, except the BUD intervention group, significantly enhanced HDAC2 activity in the mouse lung tissue (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel B). Among these, the PTX intervention group at 22.0 mg/mL demonstrated a more pronounced upregulation of HDAC2 activity, although no significant differences were observed between the groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCell Classification and Counting in BALF\u003c/h2\u003e\u003cp\u003eAfter 10 weeks of exposure to cigarette smoke and LPS, the total cell counts and the differential cell counts (including neutrophils, macrophages, lymphocytes, and eosinophils) in the BALF of the CS\u0026thinsp;+\u0026thinsp;LPS group mice were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PTX, THEO, and BUD all downregulated the expression of inflammatory cells in BALF and reduced the total cell count (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Among them, BUD demonstrated a more pronounced effect in reducing the total and individual inflammatory cell counts in BALF, although intergroup differences were minimal (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Additionally, the reduction in inflammatory cell numbers in BALF was most notable at a PTX concentration of 22.0 mg/ml, but intragroup differences remained small (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCOPD is a common, debilitating, and irreversible inflammatory lung disease closely associated with smoking. Similar to many other respiratory diseases (e.g., asthma, bronchiectasis), airway inflammation plays a crucial role in the development and progression of COPD. Chronic airway inflammation is a key driver in this process, leading to an increased presence of innate and adaptive immune cells within the airway lumen, inducing small airway fibrosis, and causing airway narrowing and eventual obstruction, accompanied by structural changes throughout the respiratory tract [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This chronic airway inflammation results from a combination of internal and environmental factors. Internal factors primarily refer to genetic susceptibility (i.e., genetic predisposition influences individual sensitivity to external factors, with specific gene polymorphisms being associated with human phenotypic responses to environmental exposure). Environmental factors include exposure to risk factors present in living and working environments, such as smoke, microorganisms, allergens like pollen and mites, air pollutants, and toxic gases [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Due to persistent nonspecific immune responses, COPD patients have increased susceptibility to infections, including respiratory viral diseases, which are among the primary triggers of AECOPD. The progression of COPD and the severity of its clinical symptoms are closely linked to chronic pulmonary inflammation. AECOPD represents an exacerbation of the inflammatory process, characterized by heightened systemic inflammatory responses, leading to disease progression, complications, and a significantly increased risk of mortality [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, effective anti-inflammatory and anti-infective treatments are essential strategies for improving the course of COPD, reducing adverse health events, and lowering the risk of mortality.\u003c/p\u003e\u003cp\u003eIt is widely acknowledged that the development and progression of COPD, as well as the severity of its clinical symptoms, are closely related to pulmonary inflammation and oxidative stress. The inflammatory response driven by an increased number of inflammatory cells is a key feature of COPD. Notably, this inflammatory response persists not only during acute exacerbations but also in stable phases of COPD [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Research has shown that 82% of acute exacerbations in COPD patients are caused by lower respiratory tract infections [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Bacterial infections exacerbate the recruitment of inflammatory cells to the lungs and increase oxidative stress, resulting in the production and release of pro-inflammatory cytokines such as reactive oxygen species (ROS), TNF-α, IL-8, IL-1, and matrix metalloproteinases (MMPs) [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. TNF-α, as a regulatory factor of inflammation, works in conjunction with chemokines to trigger an inflammatory cascade, generating more inflammatory mediators while promoting the chemotaxis of inflammatory cells within the lungs. MMP-12, in particular, sustains and amplifies the inflammatory response, further exacerbating lung inflammation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn many studies related to emphysema, \"intervention treatments\" often begin at the initial exposure of experimental animals to risk factors, before the emphysema model has developed. This approach differs from the clinical context of COPD patients, where treatment is administered after diagnosis. The former focuses on prevention, while the latter aims at treatment. Consequently, the results of such animal studies have limited relevance for guiding therapeutic strategies in patients already diagnosed with COPD [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Research has shown that intervention after the establishment of an animal disease model (\"late-stage intervention\") more accurately reflects the progression and treatment process of human diseases, offering insights into clinically relevant therapeutic strategies [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Previous studies using the \"late-stage intervention\" approach administered therapeutic drugs after cigarette smoke-induced emphysema had developed in mice and evaluated the \"treatment\" outcomes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These studies demonstrated that \"late-stage intervention\" significantly alleviated pulmonary inflammation and oxidative stress, improved the general condition of the mice, and provided valuable insights for clinical COPD management. In this experiment, we also adopted a \"late-stage intervention\" strategy. After establishing the disease model (emphysema) in experimental animals, we conducted a two-week drug intervention under the induction conditions to investigate the therapeutic effects of PTX, THEO, and BUD on experimental emphysema in mice.\u003c/p\u003e\u003cp\u003eTheophylline, a classic non-selective phosphodiesterase inhibitor (PDEI), is widely used in clinical settings for the treatment of acute asthma exacerbations. Studies have reported that theophylline at lower plasma concentrations can exhibit potential anti-inflammatory properties independent of its phosphodiesterase inhibition or antagonism of ADA receptors [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The regulation of inflammation via its effects on cAMP levels may be a key mechanism underlying its anti-inflammatory effects. In this study, theophylline was administered at a dose of 25 mg/ml. Compared to the control group, nebulized theophylline reduced the expression of IL-8, TNF-α, and IL-1β in BALF and decreased the total cell count in BALF. Similarly, Masato et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] pretreated Hartley guinea pigs sensitized with ovalbumin (OVA) with aminophylline (25 mg/ml, nebulized) and investigated the airway and inflammatory responses upon subsequent OVA exposure. Their results showed that a single dose of nebulized aminophylline could not suppress eosinophil infiltration into the airways. However, repeated nebulization (twice daily for seven days) inhibited eosinophil infiltration and reduced the total cell count and eosinophil proportion in BALF. In the present study, nebulized theophylline at 25 mg/ml in a cigarette smoke and lipopolysaccharide-induced emphysema model resulted in a decrease in BALF total cell count, consistent with the findings of Masato et al. Additionally, nebulized theophylline significantly increased HDAC2 activity. Zheng et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] demonstrated that intraperitoneal theophylline restored HDAC2 activity partially inhibited by long-term smoke exposure, significantly reduced ROS production, and showed a correlation between HDAC2 activity restoration and alleviation of oxidative stress. Thus, it is reasonable to believe that nebulized theophylline exerts anti-inflammatory effects and alleviates oxidative stress by restoring HDAC2 activity.\u003c/p\u003e\u003cp\u003ePTX is also a non-selective phosphodiesterase inhibitor (PDEI) that effectively inhibits PDE3, PDE4, and PDE7 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Studies have shown that PTX can specifically antagonize TNF-α, dose-dependently inhibiting the production of TNF-α in alveolar macrophages, as well as suppressing cytokine-induced neutrophil chemotactic factors, NF-κB, gelatinase B, and peroxidase. In inflammation, PTX exerts anti-inflammatory effects by downregulating the production of pro-inflammatory cytokines such as IL-6, IL-1, and IL-4 [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, PTX has broad antioxidant properties, and research indicates that it can alleviate oxidative stress induced by lipopolysaccharides, cigarette smoke, ozone, nitrogen mustard, and other agents [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this study, after nebulized intervention with different concentrations of PTX, the expression of TNF-α, IL-8, and IL-1β in the BALF of mice was significantly reduced. PTX also notably enhanced HDAC2 activity and decreased MMP-12 expression in lung tissues. These findings align with those of Zheng et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], despite the use of nebulized PTX in this study. To evaluate the effects of nebulized PTX on lung inflammation in smoke-exposed mice, five concentrations of PTX were tested. Although concentrations from PTX1 to PTX5 showed incremental increases, no linear relationship was observed between the concentrations and the monitored indicators. After comparing the levels of inflammatory factors in BALF, MMP-12 concentrations in lung tissues, HDAC2 activity in lung tissues, and morphological parameters, the third group (with a PTX concentration of 22.0 mg/ml) exhibited the most favorable therapeutic effects. Although the differences within the group were not statistically significant, this concentration showed better therapeutic effects compared to the others, suggesting that further animal studies are needed to determine the optimal nebulized concentration of PTX.\u003c/p\u003e\u003cp\u003eIn this study, PTX, THEO, and BUD all exhibited varying degrees of inhibition on the elevated levels of TNF-α, IL-8, IL-1β in the BALF and MMP-12 in lung tissue induced by chronic cigarette smoke and lipopolysaccharide exposure. Both PTX and THEO are phosphodiesterase inhibitors, and their effects on inflammatory factors and inflammatory cells were similar (with the exception of MMP-12, where PTX significantly downregulated its expression more than THEO). Both also had varying degrees of restoring HDAC2 activity in lung tissues. BUD, a corticosteroid commonly used in clinical anti-inflammatory treatments for conditions like AECOPD, asthma, and ARDS, significantly reduced the expression of TNF-α, IL-8, and IL-1β in BALF and the expression of MMP-12 in lung tissue. However, it did not enhance HDAC2 activity. This suggests that nebulized BUD alone does not restore HDAC2 activity. However, as mentioned in the introduction, BUD\u0026rsquo;s anti-inflammatory effects, mediated by inhibition of relevant signaling pathways, require the participation of HDAC2. Additionally, BUD is less effective in patients with stable COPD who have already experienced reduced HDAC2 activity. Since nebulized PTX and THEO significantly restore partial HDAC2 activity in lung tissues, a combination of BUD with PTX or THEO may provide better therapeutic outcomes and greater clinical benefits. However, further experiments are needed to validate this hypothesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, nebulized PTX can alleviate lung inflammation and potentially reduce oxidative stress induced by chronic cigarette smoke and lipopolysaccharide exposure, while also restoring HDAC2 activity. THEO is also able to partially restore HDAC2 activity, whereas BUD does not restore HDAC2 activity. Furthermore, within the PTX intervention group, the CS\u0026thinsp;+\u0026thinsp;LPS\u0026thinsp;+\u0026thinsp;PTX3 group (22.0 mg/ml) exhibited a trend of superior performance in reducing inflammatory factor levels in BALF, decreasing MMP-12 concentrations in lung tissue, restoring HDAC2 activity, and improving lung tissue morphology compared to other concentrations of PTX within the group, although intragroup differences were minimal (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eHowever, this study did not include roflumilast in the nebulization experiment, and due to time constraints, the combination of PTX and THEO with BUD for nebulization was not conducted. Additionally, exploring the appropriate nebulized concentration of PTX requires setting more concentration points to expand the concentration gradient. Therefore, future work should incorporate roflumilast, THEO, BUD, and PTX together in the study to observe the effects of individual nebulization versus combined nebulization with steroids. This will provide better experimental data and methodological insights for the clinical application of PTX inhalers. In conclusion, the results of this study suggest that nebulized PTX could potentially become a novel therapeutic agent for chronic inflammation in COPD, but further research is needed to determine the optimal dosage, administration methods, and the specific mechanisms of action.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (Ethics Approval Number: [2021] IACUC No. 3269). The study strictly complied with the ARRIVE Guidelines 2.0 and relevant Chinese regulations for the ethical use of laboratory animals. All procedures were designed to minimize animal suffering, including the use of appropriate anesthesia, analgesia, and humane endpoints.\u0026nbsp;The C57BL/6 mice were commercially obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). As animals were sourced from a professional laboratory animal provider and not privately owned, informed consent from owners was not required. Consent to participate is not applicable as this study utilized animal subjects.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable. This manuscript does not contain any individual person\u0026apos;s data. All experimental data derived from animal studies are published with the approval of the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (Approval ID: [2021] IACUC No. 3269).\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data underlying this article cannot be shared publicly to protect the intellectual property rights of the study funder (CSPC Pharmaceutical Group Limited) under existing contractual agreements. De-identified data will be made available to qualified researchers upon reasonable request to the corresponding author ([email protected]) after approval by both Huazhong University of Science and Technology \u0026apos;s Data Access Committee and CSPC Pharmaceutical Group Limited. Specifications of the provided paclitaxel (Pentoxifylline) are available directly from CSPC Pharmaceutical Group Limited via [email protected].\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by CSPC Pharmaceutical Group Limited. The investigational drug paclitaxel (Pentoxifylline) was provided as an in-kind contribution by the same entity.\u003c/p\u003e\n\u003cp\u003eClinical trial number\u003c/p\u003e\n\u003cp\u003enot applicable.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eHu: Conceptualization, Methodology, Investigation, Formal analysis, Writing Original Draft. Yu: Investigation, Data curation, Validation, Visualization. Peng: Resources, Software, Formal analysis. Niu: Validation, Investigation, Data curation. Li: Methodology, Resources. Wang: Supervision, Project administration. Zheng: Supervision, Project administration. Zhang*: Conceptualization, Supervision, Funding acquisition, Writing-Review \u0026amp; Editing, Project administration.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhang\u003c/strong\u003e is responsible for ensuring the accuracy and integrity of all research aspects and serves as the primary contact for communication.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the financial support provided by CSPC Pharmaceutical Group Limited for this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSAAD M I MCLEODL, HODGES C, et al. ADAM17 Deficiency Protects against Pulmonary Emphysema [J]. 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J Surg Res. 2007;143(1):99\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGHASEMNEJAD-BERENJI M, PASHAPOUR S, Pentoxifylline SADEGHPOURS. A Drug with Antiviral and Anti-Inflammatory Effects to Be Considered in the Treatment of Coronavirus Disease 2019 [J]. Med Princ Pract. 2021;30(1):98\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMINICUCCI M, OLIVEIRA F, SANTOS P, et al. Pentoxifylline Attenuates Cardiac Remodeling Induced by Tobacco Smoke Exposure [J]. Arq Bras Cardiol. 2016;106(5):396\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSUNIL V R, VAYAS K N CERVELLIJA, et al. Pentoxifylline attenuates nitrogen mustard-induced acute lung injury, oxidative stress and inflammation [J]. Exp Mol Pathol. 2014;97(1):89\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary inflammation, Cigarette smoke, Pentoxifylline, Histone deacetyltransferase 2, Cytokines","lastPublishedDoi":"10.21203/rs.3.rs-7161599/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7161599/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo explore the effect of pentoxifylline dose and administration mode on lung pathology and inflammation induced by atomization exposure of cigarettes combined with lipopolysaccharides.\u003c/p\u003e\u003ch2\u003eMetholds:\u003c/h2\u003e\u003cp\u003eFemale C57BL/6 mice were exposed to smoke (CS)\u0026thinsp;+\u0026thinsp;lipopolysaccharide (LPS) and pseudosmoke (SCS) for 10 weeks, and from week 9, animals were randomized into separate interventions with nebulized pentoxifylline (different doses), theophylline, budesonide suspension for 2 weeks, and a co-solvent control group was established. Animals are euthanized on the weekend of the 10th week. The ELISA method detected TNF-α, IL-8 and IL-1β expression in alveolar lavage fluid (BALF). After homogenization, the expression of MMP-12 and HDAC2 activity were detected by ELISA method; H\u0026amp;E staining of lung tissue sections to measure alveolar mean intercept (Lm) and alveolar destruction index (ADI); Reye-Jimsa staining assay for the determination of cell classification and quantity in BALF.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe inflammatory reaction of lung after chronic CS\u0026thinsp;+\u0026thinsp;LPS exposure is enhanced, which is manifested as TNF-α,IL-8,IL-1 β and MMP-12 increase(CS\u0026thinsp;+\u0026thinsp;LPS vs SCS: TNF-α 68.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75pg/ml vs 32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90pg/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; IL-8 45.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72pg/ml vs 15.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84pg/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; IL-1β 36.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02pg/ml vs 11.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76pg/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; MMP-12 103.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87ng/ml vs 31.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84ng/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, the HDAC2 activity decreased(CS\u0026thinsp;+\u0026thinsp;LPS vs SCS:8.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29U/ml vs 20.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60U/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Lm and ADI increased(CS\u0026thinsp;+\u0026thinsp;LPS vs SCS: Lm,45.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50um vs 24.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93um,\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ADI,51.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90% vs 6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20%,\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).Meanwhile, the total cell count in BALF augmented(CS\u0026thinsp;+\u0026thinsp;LPS vs SCS:156.20\u0026thinsp;\u0026plusmn;\u0026thinsp;18.88 10^5/ml vs 28.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50 10^5/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Budesonide suspension has no significant effect on HDAC2 activity. Different doses of pentoxifylline (PTX) and theophylline (THEO) can restore part of HDAC2 activity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eAerosol inhalation of pentoxifylline and theophylline could reduce lung inflammation induced by cigarette smoke combined with lipopolysaccharide exposure, reduce the expression of TNF-α,IL-8,IL-1β and MMP-12,and restore the decrease in HDAC2 activity induced by long-term smoke and lipopolysaccharide exposure, while inhalation of budesonide suspension alone had no effect on the activity of lung HDAC2. The recovery of HDAC2 activity is related to the nebulized inhalation dose of pentoxifylline, but more experimental studies are needed to determine the optimal concentration.\u003c/p\u003e","manuscriptTitle":"Efficacy of Pentoxifylline in Smoking Combined with Lipopolysaccharide Atomization Exposure Induced Emphysema in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 10:42:42","doi":"10.21203/rs.3.rs-7161599/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-09-07T10:40:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61977417567962851096180899161304446108","date":"2025-08-31T12:18:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-26T17:01:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310925374429032879487356519394252004552","date":"2025-08-26T16:26:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118768069080288273241288022591490936651","date":"2025-08-14T14:13:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T09:07:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T09:04:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-14T07:24:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-13T16:00:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2025-08-13T15:56:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b61146c-5839-4fd4-ab18-ddd418f3eecf","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-22T10:42:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 10:42:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7161599","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7161599","identity":"rs-7161599","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}