Cigarette Smoke Model Research Articles

Evaluation of the Effects of Locally Applied Resveratrol and Cigarette Smoking on Bone Healing

Background: Bone healing is a complex process controlled by various mechanisms. It is known that cigarette smoking (CS) negatively affects bone healing by disrupting many of these mechanisms. In an effort to find ways to eliminate these negative effects caused by CS, studies have been conducted on various vitamins, antioxidants, and medications. Since high doses and repeated injections are required to increase the therapeutic effect of conventional drug applications, controlled drug delivery systems have been developed to avoid such problems. This study aimed to investigate the effects of resveratrol (RES), which has been made into a controlled drug delivery system, on bone healing in rats that were experimentally exposed to cigarette smoke to create a chronic smoking model. Methods: After establishing a chronic CS model by exposing the subjects to cigarette smoke of six cigarettes/day for four weeks, monocortical critical size defects of 3 mm (SD ± 0.02 mm) in diameter were created in the femur using a trephine bur. During the operation, the defects in RES groups were filled locally with a gel-formed solution of RES (50 µM) and Pluronic F-127 (14 µL). CS exposure was continued during the bone healing period after surgery. All groups were sacrificed one month after the operation, and femur samples were taken. Results: The obtained samples were examined by histomorphometric and immunohistochemical techniques; osteoblast count, new bone area, macroscopic filling score, vascularization, and proliferation were evaluated. Conclusion: The results of this study indicate that CS negatively affects bone healing and that local application of RES reduces this effect.

Anti-CELA1 antibody KF4 prevents emphysema by inhibiting stretch-mediated remodeling.

There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non-AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1-/- mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix-stabilizing therapy in emphysema.

Seeds of Ginkgo biloba L. inhibit oxidative stress and inflammation induced by cigarette smoke in COPD rats through the Nrf2 pathway

Ethnopharmacological relevanceAs a traditional medicine, seeds of Ginkgo biloba L. (Gbs) have been used to treat cough or asthma for a long time. It is commonly used in clinic for lung diseases. However, its mechanism of lung protection is not completely clear. Aims of the studyThis research was designed to explore the protective effects of Gbs on antioxidant and inflammation during the chronic obstructive pulmonary disease (COPD) pathological process provoked by cigarette smoking (CS) in rats. Materials and methodsSix random groups including control group, CS model group, Gbs intervention groups (25 mg/kg, 50 mg/kg, and 100 mg/kg) and aminophylline group were composed of forty-eight rats. Smoking and intratracheal instillation of lipopolysaccharide (LPS) were used to establish the COPD rat model. Glutathione peroxidase (GSH-PX), malondialdehyde (MDA), superoxide dismutase (SOD), and enzyme-linked immunosorbent assay (ELISA) was used for quantifying the inflammatory factors such as IL-8, IL-6, IL-10, IL-17 and TNF-α. Western blotting were used for detecting the protein expressions of Nrf2, Keap1 and HO-1 in the lung tissues. ResultsGbs inhibits lung histological changes and decreased the inflammatory factors in both bronchoalveolar lavage fluid (BALF) and serum of CS-exposed rats, including IL-10, IL-17, IL-6, IL-8 and TNF-α. Gbs also inhibited the MDA level, increased SOD and GSH-PX activity in serum and changed expressions of Nrf2, Keap1 and HO-1 in the lung tissues. ConclusionGbs inhibit oxidative stress and inflammation induced by cigarette smoke in COPD rats through the Nrf2 Pathway.

Targeting the human βc receptor inhibits inflammatory myeloid cells and lung injury caused by acute cigarette smoke exposure.

Background and objectiveChronic obstructive pulmonary disease (COPD) is a devastating disease commonly caused by cigarette smoke (CS) exposure that drives tissue injury by persistently recruiting myeloid cells into the lungs. A significant portion of COPD patients also present with overlapping asthma pathology including eosinophilic inflammation. The βc cytokine family includes granulocyte monocyte‐colony‐stimulating factor, IL‐5 and IL‐3 that signal through their common receptor subunit βc to promote the expansion and survival of multiple myeloid cells including monocytes/macrophages, neutrophils and eosinophils.MethodsWe have used our unique human βc receptor transgenic (hβcTg) mouse strain that expresses human βc instead of mouse βc and βIL3 in an acute CS exposure model. Lung tissue injury was assessed by histology and measurement of albumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid. Transgenic mice were treated with an antibody (CSL311) that inhibits human βc signalling.ResultshβcTg mice responded to acute CS exposure by expanding blood myeloid cell numbers and recruiting monocyte‐derived macrophages (cluster of differentiation 11b+ [CD11b+] interstitial and exudative macrophages [IM and ExM]), neutrophils and eosinophils into the lungs. This inflammatory response was associated with lung tissue injury and oedema. Importantly, CSL311 treatment in CS‐exposed mice markedly reduced myeloid cell numbers in the blood and BAL compartment. Furthermore, CSL311 significantly reduced lung CD11b+ IM and ExM, neutrophils and eosinophils, and this decline was associated with a significant reduction in matrix metalloproteinase‐12 (MMP‐12) and IL‐17A expression, tissue injury and oedema.ConclusionThis study identifies CSL311 as a therapeutic antibody that potently inhibits immunopathology and lung injury caused by acute CS exposure.

Suhuang Antitussive Capsule Ameliorates Corticosteroid Insensitivity in Cough Variant Asthma Guinea Pigs by Inhibiting p38 MAPK Signal Pathway.

Methods The CVA guinea pig model was successfully established by use of ovalbumin (OVA) sensitization and cigarette smoke (CS) exposure. The guinea pigs were divided into 6 groups: a control group, an OVA model group, an OVA + CS model group, a Suhuang treatment group, a BUD treatment group, and a combination (Suhuang and BUD) treatment group. The effects of the treatment were determined by measuring lung function (RI/Cydn) and cough symptoms (coughs number/cough latency) as outcome criteria. The levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were determined by ELISA. Lung tissues were stained by hematoxylin and eosin (H&E). The expressions of GR/total p38 MAPK/p-p38 MAPK were detected by Western blot. The MKP-1 mRNA levels were detected by RT-PCR. Results Combination treatment significantly decreased RI/coughs numbers and increased Cydn/cough latency. Significantly, the results indicated that combination treatment decreased injury to pulmonary tissues. Results also revealed that levels of inflammatory cytokines were reduced in all treatment groups but most markedly in the combination treatment group. Moreover, Suhuang treatment significantly ameliorated corticosteroid insensitivity by improving the expression of glucocorticoid receptors (GR). The expressions of total p38 MAPK and p-p38 MAPK in lung tissue were significantly inhibited in the Suhuang and combination treatment groups. The MKP-1 mRNA levels in Suhuang and combination treatment groups were also increased significantly. Conclusion Suhuang was effective for reversing corticosteroid insensitivity by regulating the p38 MAPK signal pathway, and combining BUD and Suhuang treatment showed synergistic interactions in CVA guinea pigs. Our findings showed that this combination therapy might be a promising therapeutic agent for CVA and also clarified its underlying mechanism of action, providing a theoretical basis for clinical combination treatment with Suhuang and BUD in CVA patients.

An Improved Murine Premalignant Squamous Cell Model: Tobacco Smoke Exposure Augments NTCU-Induced Murine Airway Dysplasia.

Tobacco smoke-induced squamous cell lung cancer (SCC) develops from endobronchial dysplastic lesions that progress to invasive disease. A reproducible murine model recapitulating histologic progression observed in current and former smokers will advance testing of new preventive and therapeutic strategies. Previous studies show that prolonged topical application of N-nitroso-tris-chloroethylurea (NTCU) generates a range of airway lesions in sensitive mice similar to those induced by chronic tobacco smoke exposure in humans. To improve the current NTCU model and better align it with human disease, NTCU was applied to mice twice weekly for 4-5 weeks followed by a recovery period before cigarette smoke (CS) or ambient air (control) exposure for an additional 3-6 weeks. Despite the short time course, the addition of CS led to significantly more premalignant lesions (PML; 2.6 vs. 0.5; P < 0.02) and resulted in fewer alveolar macrophages (52,000 macrophages/mL BALF vs. 68,000; P < 0.05) compared with control mice. This improved NTCU + CS model is the first murine SCC model to incorporate tobacco smoke and is more amenable to preclinical studies because of the increased number of PML, decreased number of mice required, and reduced time needed for PML development.

Oxidative stress and mitochondrial dysfunction in parafacial respiratory group induced by maternal cigarette smoke exposure in rat offspring

Cigarette smoke (CS) exposure negatively affects neurodevelopment. We established a CS exposure rat model to determine how maternal CS exposure induces oxidative stress and mitochondrial dysfunction in parafacial respiratory group (pFRG) essential to central chemoreceptive regulation of normal breathing. Pregnant rats were exposed to CS during gestational days 1–20, and the offspring were studied on postnatal day 2. Our data showed that maternal CS exposure resulted in elevated accumulation of ROS, which left a footprint on DNA and lipid with increases in 8-hydroxy-2′-deoxyguanosine and malondialdehyde contents. Furthermore, maternal CS exposure induced decreases in manganese superoxide dismutase, catalase and glutathione reductase activities as well as reduction in glutathione content in pFRG in the offspring. Moreover, maternal exposure to CS led to mitochondrial ultrastructure changes, mitochondrial swelling, reduction in ATP generation, loss of mitochondrial membrane potential and increase in mitochondrial DNA copy number. These findings suggest that maternal exposure to CS alters normal development of pFRG that is critical for normal respiratory control.

Cigarette smoke up‐regulates PDE3 and PDE4 to decrease cAMP in airway cells

Background and PurposecAMP is a central second messenger that broadly regulates cell function and can underpin pathophysiology. In chronic obstructive pulmonary disease, a lung disease primarily provoked by cigarette smoke (CS), the activation of cAMP‐dependent pathways, via inhibition of hydrolyzing PDEs, is a major therapeutic strategy. Mechanisms that disrupt cAMP signalling in airway cells, in particular regulation of endogenous PDEs, are poorly understood.Experimental ApproachWe used a novel Förster resonance energy transfer (FRET) based cAMP biosensor in mice in vivo, ex vivo precision cut lung slices (PCLS) and in human cell models, in vitro, to track the effects of CS exposure.Key ResultsUnder fenoterol stimulation, FRET responses to cilostamide were significantly increased in in vivo, ex vivo PCLS exposed to CS and in human airway smooth muscle cells exposed to CS extract. FRET signals to rolipram were only increased in the in vivo CS model. Under basal conditions, FRET responses to cilostamide and rolipram were significantly increased in in vivo, ex vivo PCLS exposed to CS. Elevated FRET signals to rolipram correlated with a protein up‐regulation of PDE4 subtypes. In ex vivo PCLS exposed to CS extract, rolipram reversed down‐regulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre‐contracted airways.Conclusion and ImplicationsExposure to CS, in vitro or in vivo, up‐regulated expression and activity of both PDE3 and PDE4, which affected real‐time cAMP dynamics. These mechanisms determine the availability of cAMP and can contribute to CS‐induced pulmonary pathophysiology.

Establishment and Evaluation of a Rat Model of Sidestream Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a common cause of mortality worldwide. The current lack of an animal model that can be established within a certain time frame and imitate the unique features of the disease is a major limiting factor in its study. The present study established and evaluated an animal model of COPD that represents the early and advanced stage features using short-, middle-, and long-term sidestream cigarette smoke (CS) exposure. One hundred and nine Sprague–Dawley rats were randomly divided into 10 groups for different periods of sidestream CS exposure or no exposure (i.e., normal groups). The rats were exposed to CS from 3R4F cigarettes in an exposure chamber. Histological analysis was performed to determine pathological changes. We also conducted open-field tests, lung function evaluations, and cytokine analysis of the blood serum, bronchoalveolar lavage fluid, and lung tissue. The lung tissue protein levels, blood gases, and were also analyzed. As the CS exposure time increased, the indicators associated with oxidative stress, inflammatory responses, and airway remodeling were greater in the CS exposure groups than in the normal group. At 24 and 36 weeks, the COPD model rats displayed the middle- and advanced-stage features of COPD, respectively. In the 8-week CS exposure group, after the CS exposure was stopped for 4 weeks, inflammatory responses and oxidative responses were ameliorated and lung function exacerbation was reduced compared with the 12-week CS exposure group. Therefore, we established a more adequate rat model of sidestream CS induced COPD, which will have great significance for a better understanding of the pathogenesis of COPD and drug effectiveness evaluation.

Non-antibiotic 12-membered macrolides: design, synthesis and biological evaluation in a cigarette-smoking model.

The 14-membered macrolide erythromycin A expresses three distinct biological properties, including antibacterial activity, gastrointestinal motor-stimulating activity and anti-inflammatory and/or immunomodulatory effects. Although low-dose, long-term therapy using 14- and 15-membered macrolides displaying anti-inflammatory and/or immunomodulatory activity effectively treats diffuse panbronchiolitis and chronic sinusitis, bacterial resistance may emerge. To address this issue, we developed the 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900) that promotes monocyte to macrophage differentiation, a marker for anti-inflammatory and/or immunomodulatory effects, without possessing antibacterial activity. In this article, we report that the new macrolide derivative (8R,9S) -de(3'-N-methyl)-3'-N-(p-chlorobenzyl)-de(3-O-cladinosyl)-3-dehydro-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A 12,13-carbonate (EM939) exhibited stronger promotive activity for monocyte to macrophage differentiation than that of the parent compound EM900 in addition to reduced cytotoxicity toward THP-1 cells and antibacterial inactivity. In a cigarette-smoking model used to simulate chronic obstructive pulmonary disease (COPD), the EM900 derivatives significantly attenuated lung and alveolar inflations, functionally and histologically, via oral administration. Because of these marked therapeutic effects, non-antibiotic EM900 derivatives may become central to the treatment of chronic inflammatory diseases such as COPD.

Interferences in myeloid NF-κB signaling affects the inflammatory phenotype after acute smoke exposure and worsens lung damage in a murine model of cigarette smoke-induced emphysema

Cigarette smoking is the main risk factor for the chronic obstructive pulmonary disease (COPD). It was the aim of this study to examine the function of myeloid cell nuclear factor-κB (NF-κB) in cigarette smoke (CS) induced lung inflammation and lung damage. We exposed wild type mice and mice deficient for myeloid RelA/p65 or myeloid IκBα to acute or chronic CS. Numbers of macrophages were increased in RelA/p65-deficient mice and decreased in IκBα-deficient mice after acute cigarette smoke exposure. In the chronic CS model, assessment of pulmonary function showed that the total lung capacity was increased in room air and CS exposed RelA/p65- and IκBα-deficient mice. The respiratory compliance and resistance was increased and the tissue elasticity was decreased in room air exposed IκBα-deficient mice. Histology and morphometry showed that chronic exposure to CS results in loss of lung structure in RelA/p65- and IκBα-deficient mice. In conclusion, myeloid NF-κB regulates the influx of macrophages into the lung in the acute smoke setting. Myeloid NF-κB is required for maintenance of pulmonary function and lung structure and its deregulation worsens CS-induced lung damage.

Tiotropium effects on airway inflammatory events in the cat as an animal model for acute cigarette smoke-induced lung inflammation

ABSTRACTBackground: Chronic obstructive pulmonary disease is an inflammatory lung disease mainly caused by tobacco smoke inhalation. Methods: Fifteen healthy adult male cats were categorized into 3 groups: (1) control group, (2) exposed to cigarette smoke (CS), and (3) exposed to CS treated with tiotropium. Results: Increases in clinical signs and airway responsiveness in CS cats were found compared to control animals. The airway hyperresponsiveness and clinical signs were significantly attenuated by treatment with tiotropium. The CS-induced pulmonary release of interleukin-6, interleukin-8, monocyte chemotactic protein-1, and tumor necrosis factor alpha was reduced in the tiotropium group. Exposure to CS significantly increased total inflammatory cells number in bronchoalveolar lavage fluid, which was significantly attenuated by treatment with tiotropium. The number of macrophages, eosinophils and neutrophils and lymphocytes was increased after exposure to CS. Tiotropium significantly reduced the number of all these cells. Perivascular, peribronchiolar infiltration of inflammatory cells and Reid index increased in the CS group. Treatment with tiotropium significantly reduced these parameters to control level. Enhanced lipid peroxidation with concomitant reduction of antioxidants status was observed in the CS group. Tiotropium significantly reduced the serum, lung lavage, lung, and tracheal tissue lipid peroxides to near control levels. Tiotropium also decreased lung and tracheal protein leakage, and prevented the reduction of total antioxidant status in serum, lung lavage, lung and tracheal tissue of the CS group. Conclusion: Cigarette smoke increases airway responsiveness and inflammation in a cat model of CS induced lung inflammation. It can effectively be reduced by treatment with tiotropium.

Regulation of MMP-9 by a WIN-Binding Site in the Monocyte-Macrophage System Independent from Cannabinoid Receptors

The cannabinoid system is known to be involved in the regulation of inflammatory processes. Therefore, drugs targeting cannabinoid receptors are considered as candidates for anti-inflammatory and tissue protective therapy. We demonstrated that the prototypical cannabinoid agonist R(+)WIN55,212-2 (WIN) reduced the secretion of matrix metalloproteinase-9 (MMP-9) in a murine model of cigarette-smoke induced lung inflammation. In experiments using primary cells and cell lines of the monocyte-macrophage-system we found that binding of the cannabinoid-receptor agonist WIN to a stereo-selective, specific binding site in cells of the monocyte-macrophage-system induced a significant down-regulation of MMP-9 secretion and disturbance of intracellular processing, which subsequently down-regulated MMP-9 mRNA expression via a ERK1/2-phosphorylation-dependent pathway. Surprisingly, the anti-inflammatory effect was independent from classical cannabinoid receptors. Our experiments supposed an involvement of TRPV1, but other yet unidentified sites are also possible. We conclude that cannabinoid-induced control of MMP-9 in the monocyte-macrophage system via a cannabinoid-receptor independent pathway represents a general option for tissue protection during inflammation, such as during lung inflammation and other diseases associated with inflammatory tissue damage.

The Role of ChemR23 in the Induction and Resolution of Cigarette Smoke-Induced Inflammation

Chronic obstructive pulmonary disease is mainly triggered by cigarette smoke (CS) and progresses even after smoking cessation. CS induces an exaggerated influx of inflammatory cells to the bronchoalveolar space and lung parenchyma, likely resulting from a complex interplay between chemoattractants and their respective receptors. In a murine CS model of chronic obstructive pulmonary disease, we studied the importance of chemokine-like receptor ChemR23 for the induction and resolution of inflammation in CS-exposed lungs. Subacute and chronic CS exposure increased protein levels of the ChemR23 ligand and chemoattractant, chemerin, in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. Moreover, the proinflammatory chemokines CXCL1, CCL2, and CCL20 were increased in the airways of CS-exposed WT mice, accompanied by a massive accumulation of inflammatory neutrophils and monocytes, CD11b(hi)CD103(-) and CD11b(lo)CD103(+) dendritic cells (DCs), and CD4(+) and CD8(+) T cells. The lung parenchyma of WT mice was infiltrated with inflammatory neutrophils, CD11b(hi)CD103(-) DCs, and activated CD4(+) T cells after CS exposure. CS-induced inflammation was severely attenuated in BAL fluid and lungs of ChemR23 knockout mice with regard to the induction of inflammatory chemokines and the recruitment of inflammatory cells. Neutrophils and CD8(+) T cells persisted in the airways of WT mice, as did the airway-derived conventional DCs in the mediastinal lymph nodes, for at least 14 d after smoking cessation. In the BAL fluid of CS-exposed ChemR23 knockout mice, there was a remarkable delayed accumulation of T cells 14 d after the final exposure. Our data support a role for ChemR23 in directing innate and adaptive immune cells to CS-exposed lungs.

Different Inhibitory Effects of Sevoflurane on Hyperreactive Airway Smooth Muscle Contractility in Ovalbumin-sensitized and Chronic Cigarette-smoking Guinea Pig Models

The authors hypothesized that sevoflurane had different inhibitory effects on hyperreactive airway smooth muscle contractility in different types of hyperreactive airway models. The effects of sevoflurane on hyperreactive airways in ovalbumin-sensitized and chronic cigarette-smoking guinea pig models were investigated by measuring (1) total lung resistance, (2) smooth muscle tension and intracellular concentration of free Ca, (3) voltage-dependent Ca channel activity, and (4) cyclic adenosine monophosphate levels. Ovalbumin and muscarinic airway hyperreactivity was seen in ovalbumin-sensitized animals. Enlarged alveolar ducts/alveoli and lesser muscarinic hyperreactivity were observed in chronic cigarette-smoke animals. Although sevoflurane inhibited the acetylcholine-induced increase in total lung resistance in the control and ovalbumin-sensitized models, the anesthetic had a smaller effect in the chronic cigarette-smoking model. Similarly, in the chronic cigarette-smoking model, sevoflurane had a smaller inhibitory effect on carbachol-induced muscle contraction and increase in intracellular concentration of free Ca. Sevoflurane also had a smaller inhibitory effect on voltage-dependent Ca channel activity in the chronic cigarette-smoking group than in the other two groups. The sevoflurane-induced increase in cyclic adenosine monophosphate that was seen in the control and ovalbumin-sensitized groups was significantly suppressed in the chronic cigarette-smoking group. Although sevoflurane potently inhibited airway contractility in control and ovalbumin-sensitized models, the anesthetic had a smaller effect in a chronic cigarette-smoking model. The different inhibitory effects of sevoflurane on airway contractility depend, at least in part, on different effects on voltage-dependent Ca channel activity and cyclic adenosine monophosphate level.

Elevated expression of TARC (CCL17) and MDC (CCL22) in models of cigarette smoke-induced pulmonary inflammation

TARC (CCL17) and MDC (CCL22) are well-known chemoattractants for Th2 cells. Here, we evaluated the role of both chemokines for cigarette smoke-induced airway inflammation. The expression profiles of MDC, TARC, and their receptor CCR4 were analyzed in models of acute and chronic cigarette smoke-induced airway inflammation that is characterized by a Th1 immune response. The results were compared to the expression of both chemokines in models of idiopathic pulmonary fibrosis and acute asthma, which are associated with a Th2 immune response. The expression of MDC and TARC was found to be elevated in all lung inflammation models. In contrast to the findings in the asthma and lung fibrosis models, the increased expression of MDC and TARC in the cigarette-smoke model was not associated with an increased infiltration of Th2 cells into smoke-treated lungs. Our data indicate that instead of Th2 cells, airway epithelial cells expressing CCR4 might be the principal targets for MDC and TARC released from alveolar macrophages during cigarette smoke-induced airway inflammation.

Alteration of Gene Expression in Intervertebral Disc Degeneration of Passive Cigarette- Smoking Rats: Separate Quantitation in Separated Nucleus pulposus and Annulus fibrosus

Objective: We constructed a passive cigarette-smoking model with rats to investigate the molecular mechanism of intervertebral disc degeneration, and foundby gene expression analysis that passive cigarette smoking stimulated the stress-responsive signal pathway and inhibited the apoptotic pathway. In this study, to clarify that these changes were derived from either nucleus pulposus (NP) or annulus fibrosus (AF), we separately collected NP and AF and quantitatively analyzed gene expression. Methods: Total RNA was extracted from NP and AF of the lumbar intervertebral discs from rats which were kept in a smoking box for 4 and 8 weeks. Gene expression was measured by real-time PCR of cDNA synthesized from the total RNA. Results: Stress-responsive protein, heat shock protein 70, was expressed similarly in NP and AF, and wasupregulated to the same degree after 8 weeks of passive cigarette smoking. The protein tyrosine phosphatase gene was expressed more strongly in AF than in NP, and wasupregulated after 8 weeks of smoking in both tissueparts. The type II collagen and aggrecan genes were predominantly expressed in AF and NP, respectively. Conclusion: These results indicate that passive cigarette smoking stimulates both NP and AF, and induces the stress-responsible genes such as heat shock protein 70 and protein tyrosine phosphatase in both.