[The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma].

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 µg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

[Effects of caspase recruitment domain protein 9 gene knockout on airway inflammation in C57BL/6 mice with steroid resistant asthma].

Objective: To investigate the role of caspase recruitment domain protein 9 (CARD9) in airway injury and inflammation of steroid resistant asthma in C57BL/6 mice. Methods: C57BL/6 mice were divided into A group (control group), B group (model group) and C group (dexamethasone treatment group), with 6 mouse in each group using random number table. The mouse asthma model was established in B and C group by subcutaneous injection of ovalbumin (OVA)/complete Freund adjuvant (CFA) in the abdomen and OVA aerosol challenge, the pathological change and cell count in broncho alveolar lavage fluid (BALF) were detected in order to confirm the model as steroid resistant asthma, and the lung tissue inflammatory infiltration was scored. Western blot was used to detect the changes of CARD9 protein between the group A and B; then wild-type and CARD9 knockout mice were divided into D group (wild-type control group), E group (wild-type model group), F group (CARD9 knockout control group) and G group (CARD9 knockout model group), the following indicators were observed and compared after establishing steroid resistant asthma model separately: HE staining was used to observe the pathological changes of lung tissue, ELISA was used to detect the protein levels of interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-17(IL-17) in BALF, and RT-PCR was used to detect the mRNA levels of CXC motif chemokine ligand-10 (CXCL-10) and IL-17 in lung. Results: The inflammatory score (3.33±0.82 vs 0.67±0.52) and BALF total cell count [(10.13±4.83) ×105/ml vs (3.76±0.84) ×105/ml] in B group were higher than those in the A group with statistical significance (P<0.05). There was no significant difference between group C and group B in inflammatory infiltration score (2.83±0.75 vs 3.33±0.82) and BALF total cell count [(9.80±3.19) ×105/ml vs (10.13±4.83) ×105/ml] (P>0.05). Moreover the protein level of CARD9 was increased in the B group than A group (0.245±0.090 vs 0.047±0.014, P=0.004). Compared to E group and F group, more obviously inflammatory cells, neutrophils, eosinophils infiltration and tissue injury were observed in G group (P<0.05), so did the expression of IL-4 (P<0.05), IL-5 and IL-17. Meanwhile the mRNA expression levels of IL-17 and CXCL-10 also increased in lung tissue (P<0.05) of G group. Conclusion: CARD9 gene deletion may aggravate the steroid resistant of asthma by increasing neutrophil chemokines, such as IL-17 and CXCL-10, therefore increasing infiltration of neutrophils in C57BL/6 mice asthma model.

[Expression of RAGE in asthmatic rats and the intervention of Roxithromycin].

Objective: To observe the expression of the Receptor of Advanced glycation end products (RAGE) in asthmatic rats, and explore the intervention of Roxithromycin. Methods: A total of 18 Specific Pathogen Free-class Brown Norway male rats were randomly divided into control group, asthma model group and Roxithromycin group, with 6 rats in each group. The asthmatic model was sensitized by intraperitoneal injection of Ovalbumin (OVA)+Al(OH)(3), and challenged with OVA. Rats in Roxithromycin group were given Roxithromycin 30 mg/kg 30 minutes before each challenge. Rats in control group and asthma model group were treated with equal volume of saline. The concentrations of RAGE and interleukin (IL)-4 in serum and bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent (ELISA); the pathological changes of lung tissues were observed by HE-staining; the thickness of airway wall and airway smooth muscle were measured by Image-Pro Plus; the relative expression of RAGE in lung tissues were detected by Western blot. Results: In asthma model group, the concentrations of RAGE and IL-4 in the serum and BALF were obviously higher than those in control group [(494±32) vs (327±45) ng/L; (32.4±5.8) vs (13.1±2.9) ng/L; (553±38) vs (399±56) ng/L; (37.8±3.4) vs (19.4±2.5) ng/L] (all P<0.01); in Roxithromycin group, the concentrations of RAGE and IL-4 in the serum and BALF were obviously lower than those in asthma model group [(438±18) vs (494±32) ng/L; (22.8±6.0) vs (32.4±5.8) ng/L; (444±42) vs (553±38) ng/L; (25.6±4.5) vs (37.8±3.4) ng/L] (all P<0.05). In asthma model group, the bronchial wall was thickened, the lumen was narrow, the mucosal wrinkles were significantly increased, edema appeared under the mucosa, and a large number of inflammatory cells infiltrated and aggregated in the bronchi, perivascular and alveolar spaces; the thickness of airway wall and airway smooth muscle were significantly increased than those in control group (P<0.01); in Roxithromycin group, airway inflammation and remodeling were alleviated compared with those in asthma model group (P<0.05). In asthma model group, the expression of RAGE in lung tissues were significantly increased than those in control group (P<0.01); in Roxithromycin group, the expression of RAGE were significantly decreased than those in asthma model group (P<0.01). There were positive correlations between the expression of RAGE and IL-4 in BALF and serum (r=0.782, 0.804, all P<0.01); there were positive correlations between RAGE and total white cell counts, eosinophil counts, smooth muscle thickness (r=0.897, 0.927, 0.860, all P<0.01). Conclusions: The increasing of RAGE in asthmatic rats are positively correlated with airway inflammation and airway remodeling. Roxithromycin may inhibit the development of asthma by reducing the expression of RAGE.

[The role of S100A8/RAGE and Caveolin-1 and the effect of roxithromycin on their expression in a rat model of neutrophilic asthma].

Objective: To explore the role of S100A8, the receptor for advanced glycation endproducts (RAGE) and Caveolin-1 in neutrophilic asthmatic rats, and to further study the intervention of roxithromycin and the possible mechanisms. Methods: Male Brown Norway rats were randomly assigned to a control group, an asthma group and a Roxithromycin group. The asthmatic rat model was established by intraperitoneal injection of ovalbumin (OVA) and Freund's complete adjuvant (FCA) mixture, and aerosol inhalation of OVA. Rats in the Roxithromycin group were given roxithromycin injection 30 mg/kg 30 minutes before each challenge. Rats in the control and the asthma groups were replaced with equal volumes of saline, respectively. Bronchoalveolar lavage fluid (BALF) neutrophil percentage (Neu%) and pathological changes of pulmonary tissue (hematoxylin-eosin, HE staining) were measured to confirm the establishment of asthmatic models. The concentration of inflammatory cytokines and S100A8 were quantified by enzyme-linked immunosorbent assay (ELISA), and the expression of Caveolin-1 and RAGE at protein levels were detected by immunohistochemistry and Western blot. Results: Neu% in BALF of the asthma group was significantly higher than those of the control group, and Neu% in the Roxithromycin group was lower than the asthma group (all P<0.01). Pulmonary histology revealed that there were a large number of inflammatory cells infiltrated in the bronchial and perivascular, pulmonary interstitial and alveolar spaces, and the bronchial wall and smooth muscles were thickened obviously in the asthma group. Rats in the Roxithromycin group showed milder inflammation and airway remodeling change than the asthma group. There was no obvious pathological damage in the control group. The concentration of IL-6 and IL-17 in BALF and serum of rats in the asthma group were significantly higher than those in the control group (P<0.01), and Roxithromycin inhibited the high expression of these cytokines (P<0.05). The expression of S100A8 and RAGE in the asthma group were significantly higher than those in the control group [(20.6±4.4) vs (7.1±2.0) ng/L; (885±118) vs (462±102) ng/L; (14.2±1.7) vs (7.6±1.8) ng/L; (774±166) vs (406±69) ng/L, all P<0.05], and Roxithromycin inhibited the high expression of these proteins [(14.3±3.7) vs (20.6±4.4) ng/L; (650±53) vs (885±118) ng/L; (10.4±1.2) vs (14.2±1.7) ng/L; (560±64) vs (728±72) ng/L] (all P<0.05). Meanwhile, the expression of Caveolin-1 in the asthma group was significantly lower than that in the control group (P<0.01), and Roxithromycin up-regulated its expression (P<0.01). Correlation analysis showed that there was a significantly positive correlation between the expression of S100A8 and RAGE (r=0.706, P<0.01), while there was a significantly negative correlation between the expression of S100A8 and Caveolin-1 (r=-0.775, P<0.01), and between the expression of Caveolin-1 and RAGE (r=-0.919, P<0.01). Conclusion: S100A8 and Caveolin-1 may play an important role in neutrophilic asthma via RAGE, and Roxithromycin may exerts anti-inflammatory effects and inhibition of airway remodeling partly through this signaling pathway.

[Chinese expert consensus for non-antiinfective effects and clinical use of macrolides].

Important/potential value of macrolides has been proved in the management of chronic respiratory diseases by increasing basic and clinical trials.Through three face-to-face discussions, 10 experts examined important data and drafted this consensus related to macrolides: (1) mechanism of non-antiinfective effects; (2) clinical use in chronic respiratory diseases; (3) cautions of long-term use.The mechanism out of non-antiinfective effects includes anti-inflammatory effect, modifying airway secretion, immune-regulation related to antibacterial effect, corticoid saving effect and anti-viral effect.The efficacy of long-term use of low-dose macrolides is definitely confirmed in diffuse panbronchiolitis, chronic rhinosinusitis. It is considerably used in bronchiectasia, cystic fibrosis, severe asthma and chronic obstructive pulmonary disease. Further studies should be conducted in cryptogenic organizing pneumonia and respiratory viral infection. It should be paid attention to its possible adverse effects (including drug interactions, cardiac toxicity, ototoxicity and disturbance of intestinal flora) and drug resistance in long-term use.A Chinese consensus for non-antiinfective effects and clinical use of macrolides is developed for the first time, which aims to expand their rational use and the further research.

[Role of roxithromycin on glucocorticoid resistance of human bronchial epithelial cells and its mechanism].

Objective: To explore the effects of roxithromycin (RXM) on glucocorticoid resistance of human bronchial epithelial cells exposed to smoke and its mechanism. Methods: Beas-2B cells as the research object were grouped into: control group, 10%cigarette smoke extract (CSE) group, roxithromycin (RXM)+ 10%CSE group. With 10%CSE intervention in the 10%CSE group, 10%CSE and RXM intervention in the RXM+ 10%CSE group, complete culture solution intervention in the control group. Interleukin-8 (IL-8) levels were measured by enzyme linked immunosorbent assay (ELISA) and IL-8 inhibition rate and dexamethasone half inhibitory concentration (IC50-Dex) were calculated; the expression of histone deacetylase 2 (HDAC2) protein was detected by immunofluorescence (IF) and Western blotting (WB). Results: In response to dexamethasone at the concentration of 10(-9,) 10(-8,) 10(-7) and 10(-6) mol/L successively, the IL-8 inhibition rates of RXM+ 10%CSE group [(27.55±3.81)%, (49.60±1.45)%, (55.36±3.36)%, (60.32±3.13)%, respectively] were lower than those of control group [(32.85±2.56)%, (57.12±2.81)%, (60.81±2.08)%, (67.24±3.50)%, respectively], but higher than those of 10%CSE group [(19.15±1.69)%, (37.02±2.30)%, (47.15±2.01)%, (52.09±1.57)%, respectively] (all P<0.05). In contrast, the IC50-Dex of RXM+ 10%CSE group [(4.94±1.62)×10(-8)] was significantly higher than that of control group [(1.75±0.77)×10(-8)], but lower than that of 10%CSE group [(2.92±0.78)×10(-7)] (both P<0.01). The expression of HDAC2 protein of 10%CSE group (0.011±0.004 from IF and 0.46±0.10 from WB) was lower than that of control group (0.037±0.005 and 0.91±0.06, correspondingly), while RXM+ 10%CSE group (0.025±0.005 and 0.77±0.09, correspondingly) was lower than that of control group but higher than that of 10%CSE group (all P<0.05). Conclusion: Roxithromycin may restrain tobacco smoke exposure-induced glucocorticoid resistance in human bronchial epithelial cells through upregulating HDAC2 expression.

[Outcomes of same-day bilateral endoscopic tympanoplasty].

Objective:To investigate the feasibility of simultaneous bilateral endoscopic for tympanoplasty in patients with bilateral chronic suppurative otitis media. Method:Fifteen patients (30 ears) with bilateral chronic suppurative otitis media who underwent bilateral endoscopic transcanal tympanoplasty on the same day were enrolled in this study. The ear with worse-hearing was selected as the first operation side, the contralateral ear as the second one. The operation group consisted of 22 ears of Type 1 tympanoplasty, 5 ears of Type 2 tympanoplasty and 3 ears of Type 3 tympanoplasty. All second sides(15 ears) underwent Type 1 tympanoplasty. The cartilage-perichondrium graft was harvested from the tragal of the first side and was cut in two halves which could be used for the both sides. The graft success and hearing improvement were evaluated at the postoperative 6th month according to the follow up results of the endoscopic image and the pure-tone audiometry. Result:The graft take rate was 96.7%(27/30) without any retraction pockets or displaced grafts. The graft take rate of the first side was 93.3%(14/15), and the one of the second side was 100.0%(15/15). The average air conduction thresholds were (50.9±9.1) dB HL preoperatively and (32.0±6.0) dB HL postoperatively(P<0.01). The average air-bone gap overall improved from (30.2±7.9) dB HL preoperatively to (13.7±6.0) dB HL postoperatively(P<0.01). Conclusion:Bilateral same-day endoscopic tympanoplasty are safe and cost effective in appropriately selected patients. It can offer favorable out-comes in selected patients with chronic suppurative otitis media.

Cleavage of lamin-like proteins in in vivo and in vitro apoptosis of tobacco protoplasts induced by heat shock.

Apoptosis in heat shock-treated tobacco protoplasts was evidenced by DNA fragmentation, flow cytometric analysis and activation of caspase 3-like protease. Furthermore, an in vitro apoptosis system was established which reproduced the apoptotic events. Western blotting analysis using an antibody against lamin A and C showed that in both in vivo and in vitro systems lamin-like proteins were cleaved into a 35-kDa fragment, and that lamin-like protein degradation precedes DNA fragmentation. Moreover, we found a 22.8-fold increase in caspase 6-like activity in cytosol of heat-treated protoplasts as compared with the control.

Hydroxyl radical-induced apoptosis in human tumor cells is associated with telomere shortening but not telomerase inhibition and caspase activation.

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as *OH and telomerase/telomere. To address the interrelations between *OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with *OH radicals generated via Fe(2+)-mediated Fenton reactions (0.1 mM FeSO(4) plus 0.3-0.9 mM H2O2) and telomerase activity, telomere length were measured during apoptosis. We found that during *OH-induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that *OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of *OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during *OH-induced apoptosis. Strikingly, we also found that *OH-induced apoptosis in HeLa cells is caspase-3-independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that *OH triggers apoptotic tumor cell death through a telomere-related, caspase-independent pathway.

Involvement of poly(ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock-induced apoptosis in tobacco suspension cells.

The cleavage of poly(ADP-ribose) polymerase (PARP) by caspase (casp)-3 is an essential link in the apoptotic pathway in animal cells. In plant cells, however, there is no authentic evidence for the similar role that PARP may play during apoptosis. Using a heat shock (HS)-induced apoptosis system of tobacco cells, we found that immediately after a 4 h heat treatment, PARP was cleaved to form an 89 kDa signature fragment, while DNA laddering appeared only after a 20 h recovery following the HS. An activation of casp-3-like protease was also observed. The results suggest that apoptosis in plants and animals may share common mechanisms. On the other hand, when cells were preincubated with 4 mM 3-aminobenzamide or 2-8 mM nicotinamide, the specific inhibitors of PARP, before HS treatment, apoptotic cell death was reduced significantly. Our results thus imply that PARP may also be involved in apoptosis in a different way from the casp-related events.

Expression of telomerase inhibits hydroxyl radical-induced apoptosis in normal telomerase negative human lung fibroblasts.

In tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase-expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical-induced apoptosis in telomerase-expressing and control fibroblasts was caspase-3 independent. These findings have revealed a new type of interrelation between telomerase and caspase-3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase-3 cascade.

2- and 6-methyl-1,4-naphthoquinone derivatives and potential bioreductive alkylating agents.

A number of antineoplastic agents possess both the quinone nucleus and an appropriate substituent that permits them to function as bioreductive alkylating agents. To develop new compounds of this type with unique properties, we have synthesized a series of 2- and 6-methyl-1,4-naphthoquinone derivatives and have evaluated them for antineoplastic activity against Sarcoma 180 ascites cells. Several of these quinones showed antitumor activity, causing significant prolongation of the survival time of tumor-bearing mice. Among the most active agents were the mesylates, tosylates, and N-(chloroethyl)carbamates of 2- and 6-methyl-1,4-naphthoquinone. That bioreductive activation to a quinone methide might be involved in the mechanism of action of these agents was shown by the finding that compounds with the best leaving groups were the most efficacious as antineoplastic agents.

[Immunocytochemical and morphologic features of poorly differentiated adenocarcinoma of the nasopharynx].

Poorly differentiated adenocarcinoma of the nasopharynx is not rare. It comprises 5% of all nasopharyngeal carcinomas. In this paper, specimens of 41 cases of poorly differentiated adenocarcinoma of the nasopharynx were studied. The microscopic findings have the tendency to form glandular or duct-like structures, or a specific "cerebriform" appearance, AB-PAS stain was positive. In addition to the common features of adenocarcinoma (cancer cells vary in size, with large, round central nuclei, enlarged conspicuous nucleoli), a specific feature that the nuclei of cancer cells were 1-2 times larger than those of normal cells was seen in smear. Electron microscopic observation revealed that the cytoplasm of the cancer cells contained numerous mitochondria, RER, developed Golgi apparatus and some secretory granules. Immunocytochemical studies proved that it was moderately positive for immunostain of low molecular weight keratin protein (K10,11), but was negative for keratin (K) it is different from poorly differentiated squamous cell carcinoma and vesicular nuclear cell carcinoma, of which were strongly positive or partially positive for keratin. The main points of differential diagnosis for these carcinomas are elucidated.

[Immunohistochemical study on differential diagnosis between NPC and malignant lymphoma].

Nasopharyngeal carcinoma (NPC) and malignant lymphoma are common malignant tumors which frequently involve nasopharynx and cervical lymph nodes. Sometimes, it is difficult to distinguish poorly-differentiated NPC, especially undifferentiated NPC, from malignant lymphoma. Paraffin sections of 221 cases of poorly differentiated or undifferentiated NPC and malignant lymphomas were analysed by immunohistochemical techniques (IGSS, ABC, double stain, etc.), The immunohistochemical criteria of differential diagnosis between NPC and malignant lymphomas were proposed and with these criteria, 40 cases which were difficult to distinguish between NPC and malignant lymphoma were identified. In comparison with the methods of SPA, PAP, ABC, IGSS, etc., and the probes of Ke, EMA, LCA, Vi, etc. on paraffin sections, IGSS or ABC method and probes of Ke and LCA were considered to be more sensitive.

[Predictive impact on malaria prevalence by hydraulic project of three gorges of the Yangtze River].

Malaria had a wide distribution and high prevalence throughout the Three Gorges region of the Yangtze River. After antimalarial program, its incidence rate was reduced to 2.2% in 1985, but sporadic cases still occurred in 34.7% of the townships and local outbreaks were not uncommon. Therefore, potential factors of malaria outbreak are still present. It is predicted that maximum risk of malaria outbreak will take place, during construction and after the completion of the reservoir, the breeding sites of Anopheles would be extended to irrigation network, low-lying of flooded land, and malaria prevalence might be increased.

Roxithromycin reduces the viability of cultured airway smooth muscle cells from a rat model of asthma.

OBJECTIVES: The purpose of this study was to investigate the effect of roxithromycin on apoptosis of airway smooth muscle cells (ASMCs) from a rat model of asthma and uncover signaling pathway underlying the cytotoxicity of roxithromycin. MATERIALS AND METHODS: ASMCs were isolated from a rat model of asthma and treated with or without roxithromycin for 48 h before parameter detection. Cell viability was assessed by WST-8 assay and flow cytometry after Annexin V/PI double staining. Changes in the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry using JC-1. Cytochrome C (Cyt c), cleaved Caspase-9/3 and P27 were evaluated by Western Blot. RESULTS: Incubation with roxithromycin reduced ASMCs proliferation and enhanced apoptosis in a dose-dependent manner. Flow cytometry revealed a loss of ΔΨm and Western Blot displayed Caspase-9/3 activation as well as Cyt c release from mitochondria to the the cytosol after the treatment of roxithromycin. In addition, P27 were more strongly expressed in AMSCs treated with roxithromycin compared with the control group. CONCLUSIONS: Roxithromycin induced apoptosis of ASMCs derived from a rat model of asthma in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway, involving the up-regulation of P27.