Biological role of mitochondrial TLR4-mediated NF-κB signaling pathway in central nervous system injury.

Previous studies suggested that central nervous system injury is often accompanied by the activation of Toll-like receptor 4/NF-κB pathway, which leads to the upregulation of proapoptotic gene expression, causes mitochondrial oxidative stress, and further aggravates the inflammatory response to induce cell apoptosis. Subsequent studies have shown that NF-κB and IκBα can directly act on mitochondria. Therefore, elucidation of the specific mechanisms of NF-κB and IκBα in mitochondria may help to discover new therapeutic targets for central nervous system injury. Recent studies have suggested that NF-κB (especially RelA) in mitochondria can inhibit mitochondrial respiration or DNA expression, leading to mitochondrial dysfunction. IκBα silencing will cause reactive oxygen species storm and initiate the mitochondrial apoptosis pathway. Other research results suggest that RelA can regulate mitochondrial respiration and energy metabolism balance by interacting with p53 and STAT3, thus initiating the mitochondrial protection mechanism. IκBα can also inhibit apoptosis in mitochondria by interacting with VDAC1 and other molecules. Regulating the biological role of NF-κB signaling pathway in mitochondria by targeting key proteins such as p53, STAT3, and VDAC1 may help maintain the balance of mitochondrial respiration and energy metabolism, thereby protecting nerve cells and reducing inflammatory storms and death caused by ischemia and hypoxia.

Value of inversion imaging to diagnosis in differentiating malignant from benign breast masses.

BACKGROUND: We aimed to evaluate the added value of inversion imaging in differentiating between benign and malignant breast masses when combined with the Breast Imaging Reporting and Data System (BI-RADS). METHODS: A total of 364 patients with 367 breast masses (151 benign and 216 malignant) who underwent conventional ultrasound and inversion imaging prior to breast surgery were included. A 5-point inversion score (IS) scale was proposed based on the masses' internal echogenicity and distribution characteristics in the inversion images. The combination of IS and BI-RADS was compared with BI-RADS alone to evaluate the value of inversion imaging for breast mass diagnosis. The diagnostic performance of the BI-RADS and its combination with IS for breast masses were analyzed using area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS: The IS for malignant breast masses (3.96 ± 0.77) was significantly higher than benign masses (2.58 ± 0.98) (P < 0.001). The sensitivity, specificity, accuracy, PPV, and NPV of BI-RADS were 86.1%, 81.5%, 84.2%, 86.9%, and 80.4%, respectively, and an AUC was 0.909. By compared with BI-RADS, 72 breast masses were downgraded from suspected malignancy to benign, and 6 masses were upgraded from benign to suspected malignancy. Thus, the specificity was increased from 81.5 to 84.8%, it allows 72 benign masses avoid biopsy. CONCLUSION: The combination of inversion imaging with BI-RADS can effectively improve the diagnostic efficacy of breast masses, and inversion imaging could help benign masses avoid biopsy.

Inhibitory mechanism of vortioxetine on CYP450 enzymes in human and rat liver microsomes.

Vortioxetine is a novel anti-major depression disorder drug with a high safety profile compared with other similar drugs. However, little research has been done on drug-drug interactions (DDI) about vortioxetine. In this paper, the inhibitory effect of vortioxetine on cytochrome P450 (CYP450) and the type of inhibitory mechanism were investigated in human and rat liver microsomes. We set up an in vitro incubation system of 200 µL to measure the metabolism of probe substrates at the present of vortioxetine at 37°C. The concentrations of the metabolites of probe substrates were all measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. It was found no time-dependent inhibition (TDI) of vortioxetine through determination of half-maximal inhibitory concentration (IC50) shift values. The enzymes and metabolites involved in this experiment in human and rats were as follows: CYP3A4/CYP3A (midazolam); CYP2B6/CYP2B (bupropion); CYP2D6/CYP2D (dextromethorphan); CYP2C8/CYP2C-1 (amodiaquine); CYP2C9/CYP2C-2 (losartan); and CYP2C19/CYP2C-3 (mephenytoin). We found that vortioxetine competitively inhibited CYP2C19 and CYP2D6 in human liver microsomes (HLMs) with inhibition constant (Ki) values of 2.17 µM and 9.37 µM, respectively. It was noncompetitive inhibition for CYP3A4 and CYP2C8, and its Ki values were 7.26 µM and 6.96 µM, respectively. For CYP2B6 and CYP2C9, vortioxetine exhibited the mixed inhibition with Ki values were 8.55 µM and 4.17 µM, respectively. In RLMs, the type of vortioxetine inhibition was uncompetitive for CYP3A and CYP2D (Ki = 4.41 and 100.9 µM). The inhibition type was competitive inhibition, including CYP2B and CYP2C-2 (Ki = 2.87 and 0.12 µM). The inhibition types of CYP2C-1 and CYP2C-3 (Ki = 39.91 and 4.23 µM) were mixed inhibition and noncompetitive inhibition, respectively. The study of the above mechanism will provide guidance for the safe clinical use of vortioxetine so that the occurrence of DDI can be avoided.

In vitro assessment of 24 CYP2D6 allelic isoforms on the metabolism of methadone.

CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme super family, with at least 100 CYP2D6 alleles being previously identified. Genetic polymorphisms of CYP2D6 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to clarify the catalytic activities of 24 CYP2D6 alleles on the oxidative in vitro metabolism of methadone. Reactions were incubated with 50-2000 µM methadone for 30 min at 37 °C and terminated by cooling to -80 °C immediately. Methadone and the major metabolite EDDP were analyzed by an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) system. Compared with wild-type CYP2D6*1, most variants showed significantly altered values in Vmax and intrinsic clearance (Vmax /Km ). Only three variants (CYP2D6*88, *91 and E215K) exhibited markedly increased intrinsic clearance values, and one variant CYP2D6*94 showed no significant difference. On the other hand, the kinetic parameters of two CYP2D6 variants (CYP2D6*92 and *96) could not be determined because they had no detectable enzyme activity, whereas 18 variants exhibited significantly decreased values. To sum up, this study demonstrated that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2D6 alleles. Copyright © 2016 John Wiley & Sons, Ltd.

Role of cytochrome P450 2D6 genetic polymorphism in carvedilol hydroxylation in vitro.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that catalyzes the metabolism of a great number of therapeutic drugs. Up to now, >100 allelic variants of CYP2D6 have been reported. Recently, we identified 22 novel variants in the Chinese population in these variants. The purpose of this study was to examine the enzymatic activity of the variants toward the CYP2D6 substrate carvedilol in vitro. The CYP2D6 proteins, including CYP2D6.1 (wild type), CYP2D6.2, CYP2D6.10, and 22 other novel CYP2D6 variants, were expressed from insect microsomes and incubated with carvedilol ranging from 1.0 µM to 50 µM at 37°C for 30 minutes. After termination, the carvedilol metabolites were extracted and detected using ultra-performance liquid chromatography tandem mass-spectrometry. Among the 24 CYP2D6 variants, CYP2D6.92 and CYP2D6.96 were catalytically inactive and the remaining 22 variants exhibited significantly decreased intrinsic clearance values (ranging from ~25% to 95%) compared with CYP2D6.1. The present data in vitro suggest that the newly found variants significantly reduced catalytic activities compared with CYP2D6.1. Given that CYP2D6 protein activities could affect carvedilol plasma levels, these findings are greatly relevant to personalized medicine.

Effects of 22 CYP2D6 Genetic Variations Newly Identified in Chinese Population on Olanzapine Metabolism in vitro.

The objective of this study was to assess the catalytic activity of 22 novel CYP2D6 allelic variants (2D6*87-*98, R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C and R497C) to olanzapine in vitro. Their protein products expressed in Spodoptera frugiperda 21 (Sf21) insect cells were incubated with olanzapine 100-2,000 µmol/l for 30 min. The kinetic parameters of Km, Vmax and intrinsic clearance were determined by 2-hydroxymethylolanzapine, the metabolite of olanzapine mediated by CYP2D6, using ultra-performance liquid chromatography tandem mass spectrometry. Results showed that the kinetic parameters of 2 alleles, CYP2D6*92 and 2D6*96, could not be detected; 17 allelic variants, CYP2D6*87-*88, 2D6*90-*91, 2D6*93-*95, 2D6*97, R25Q, F164L, E215K, F219S, V327M, V342M, R344Q, R440C and R497C, significantly reduced the intrinsic clearance of olanzapine; 2 variants, CYP2D6*89 and 2D6*98, increased the intrinsic clearance of olanzapine; no difference was found in intrinsic clearance of D336N. Furthermore, 6 alleles, CYP2D6*87, 2D6*88, 2D6*91, 2D6*93, 2D6*97 and R497C, exhibited higher Km values in a range of 120.80-217.56% relative to wild-type CYP2D6*1. The research demonstrated the metabolic phenotype of the 22 novel CYP2D6 variants for olanzapine that were different from probe drugs we used previously and might provide beneficial information to the personalized medicine of olanzapine.

Assessment of 25 CYP2D6 alleles found in the Chinese population on propafenone metabolism in vitro.

Cytochrome P450 enzyme 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily, with more than 100 CYP2D6 allelic variants being previously reported. The aim of this study was to assess the catalytic characteristics of 25 alleles (CYP2D6.1 and 24 CYP2D6 variants) and their effects on the metabolism of propafenone in vitro. Twenty-five CYP2D6 alleles were expressing in 21 Spodoptera frugiperda (Sf) insect cells, and each variant was evaluated using propafenone as the substrate. Reactions were performed at 37 °C with 1-100 µmol/L propafenone for 30 min. After termination, the product 5-OH-propafenone was extracted and used for signal collection by ultra-performance liquid chromatography (UPLC). Compared with wild type CYP2D6.1, the intrinsic clearance (Vmax and Km) values of all variants were significantly altered. Three variants (CYP2D6.87, CYP2D6.90, CYP2D6.F219S) exhibited markedly increased intrinsic clearance values (129% to 165%), whereas 21 variants exhibited significantly decreased values (16% to 85%) due to increased Km and (or) decreased Vmax values. These results indicated that the majority of tested alleles had significantly altered catalytic activity towards propafenone hydroxylation in this expression system. Attention should be paid to subjects carrying these rare alleles when treated with propafenone.

Effect of 24 cytochrome P450 2D6 variants found in the Chinese population on the N-demethylation of amitriptyline in vitro.

CONTEXT: Amitriptyline (AT), one of the tricyclic antidepressants, is still widely used for the treatment of the depression and control of anxiety states and panic disorders in the developing countries. OBJECTIVE: This study evaluates the catalytic activities of CYP2D6*1, CYP2D6*2, CYP2D6*10 and 22 novel alleles in Han Chinese population and their effects on the N-demethylation of AT in vitro. MATERIALS AND METHODS: CYP2D6*1 and 24 CYP2D6 allelic variants were highly expressed in insect cells, and all variants were characterized using AT as a substrate. Reactions were performed at 37 °C with 10-1000 µM substrate for 30 min. We established a HPLC method to quantify the levels of nortriptyline (NT). The kinetic parameters Km, Vmax and intrinsic clearance (Vmax/Km) of NT were calculated. RESULTS: Among the 24 CYP2D6 variants, all variants exhibited decreased intrinsic clearance values compared with wild-type CYP2D6.1. Kinetic parameters of two CYP2D6 variants (CYP2D6*92, *96) could not be determined because of absent enzyme activities. CONCLUSIONS: The comprehensive in vitro assessment of CYP2D6 variants provides significant insight into allele-specific activity towards AT in vivo.

Effect of CYP2D6 genetic polymorphism on the metabolism of citalopram in vitro.

Genetic polymorphisms of CYP2D6 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. We aimed at investigating the role of CYP2D6 in the metabolism of citalopram and identifying the effect of 24 CYP2D6 allelic variants we found in Chinese Han population on the metabolism of citalopram in vitro. These CYP2D6 variants expressed by insect cells system were incubated with 10-1000 µM citalopram for 30 min at 37 °C and the reaction was terminated by cooling to -80 °C immediately. Citalopram and its metabolites were analyzed by high-performance liquid chromatography (HPLC). The intrinsic clearance (Vmax/Km) values of the variants toward citalopram metabolites were significantly altered, 38-129% for demethylcitalopram and 13-138% for citalopram N-oxide when compared with CYP2D6*1. Most of the tested rare alleles exhibited significantly decreased values due to increased Km and/or decreased Vmax values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings suggest that more attention should be paid to subjects carrying these CYP2D6 alleles when administering citalopram in the clinic.

The effect of resveratrol on pharmacokinetics of aripiprazole in vivo and in vitro.

1. The objective of this study were to investigate the effect of orally administered resveratrol on the pharmacokinetics of aripiprazole (APZ) in rat, and the inhibitory effects of resveratrol on APZ dehydrogenation activity in liver microsomes and human cytochrome P450 3A4 and 2D6. 2. Twenty-five healthy male Sprague-Dawley rats were randomly divided into five groups: A (control group), B (multiple dose of 200 mg/kg resveratrol), C (multiple dose of 100 mg/kg resveratrol), D (a single dose of 200 mg/kg resveratrol) and E (a single dose of 100 mg/kg resveratrol). A single dose of 3 mg/kg APZ administered orally 30 min after administration of resveratrol. In addition, CYP2D6*1, CYP3A4*1, human and rat liver microsomes were performed to determine the effect of resveratrol on the metabolism of APZ in vitro. 3. The multiple dose of 200 or 100 mg/kg resveratrol significantly increased the AUC and Cmax of APZ. The resveratrol also obviously decreased the CL, but without any significant difference on t1/2 in vivo. On the other hand, resveratrol showed inhibitory effect on CYP3A4*1, CYP2D6*1, human and rat microsomes, the IC50 of resveratrol was 6.771, 87.87, 45.11 and 35.59 µmol l(-1), respectively. 4. Those results indicated more attention should be paid when APZ was administrated combined with resveratrol.

Effect of CYP2D6 variants on venlafaxine metabolism in vitro.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 µM substrate (three variants was adjusted to 1000 µM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.

Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high-performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1'-hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O-demethylation than those of the wild-type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild-type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.

Effect of 24 Cytochrome P450 2D6 Variants Found in the Chinese Population on Atomoxetine Metabolism in vitro.

OBJECTIVE: The aim of this article was to assess the catalytic activities of 24 cytochrome P450 2D6 (CYP2D6) variants found in the Chinese population toward atomoxetine in vitro as well as CYP2D6.1. METHODS: In this study, the co-expression enzyme of human recombinant CYPOR, CYPb5, and CYP2D6.1 or other CYP2D6 variants with the baculovirus-mediated insect cells (Sf21) was used to study the catalytic activities of 24 CYP2D6 variants toward atomoxetine metabolism. The metabolite of atomoxetine (4-hydroxyatomoxetine) was detected by ultra-high performance liquid chromatography-mass spectrometry method. RESULTS: The intrinsic clearance (Vmax/Km) values of most variants were significantly altered when compared with CYP2D6.1. CYP2D6.94, CYP2D6.D336N, CYP2D6.R440C exhibited marked increased values 172, 126, 121% respectively. CYP2D6.89 and CYP2D6.98 exhibited similar catalytic activity as the wild type, whereas 17 variants exhibited significantly decreased values (from 5 to 87%) due to increase Km and/or decrease Vmax values. However, CYP2D6.92 and CYP2D6.96 showed no or few activity because of producing nothing. CONCLUSIONS: Our results suggest that most of these newly found variants exhibit significantly changed catalytic activities compared with the wild type. And these findings provide valuable information for the growth and development of personalized medicine in China.

Effect of 22 Novel Cytochrome P450 2D6 (CYP2D6) Variants Found in the Chinese Population on Hemangeol Metabolism In Vitro.

BACKGROUND AND OBJECTIVES: Hemangeol, approved for the treatment of proliferative infantile hemangiomas requiring systemic therapy, is metabolized by cytochrome P450 2D6 (CYP2D6), which is a highly polymorphic enzyme that metabolizes a large number of drugs. More than 100 CYP2D6 allelic variants have been reported so far, including 22 novel variants that discovered in our lab in the Chinese population. Our study aimed to probe the enzymatic activity of these variants toward hemangeol in vitro with recombinant microsomes that expressed in sf21 insect cells using a baculovirus-mediated expression system. METHODS: The wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) were incubated with 1-200 µM hemangeol for 50 min at 37 °C. Then the products were extracted, and signal detection was performed by high-performance liquid chromatography with fluorescence detector. RESULTS: All of the variants exhibited changed apparent Michaelis-Menten constant (Km) or maximum velocity of the reaction (V max) values compared with that of wild-type protein. The intrinsic clearances (V max /Km) were significantly decreased by 0.37 to 42.74 %. However, CYP2D6.92 and CYP2D6.96 showed no or minimal enzymatic activity as no concentration of 4'-hydroxypropranolol was detected. CONCLUSIONS: The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards hemangeol in vivo.

Effects of 24 CYP2D6 Variants Found in the Chinese Population on the Metabolism of Risperidone.

AIMS: Cytochrome P450 (CYP450) 2D6 is an important member of the P450 enzyme superfamily and responsible for clearing 25% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 24 CYP2D6 allelic isoforms found in the Chinese population and their effects on the metabolism of risperidone in vitro. METHODS: Insect microsomes expressing wild-type CYP2D6 and 24 CYP2D6 allelic variants were incubated with 20-1,000 µmol/l risperidone for 40 min at 37°C. After termination, risperidone and 9-OH risperidone, the metabolite of risperidone, were precipitated and used for signal collection by ultra-performance liquid-chromatography tandem mass spectrometry. RESULTS: Among 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be with no detectable activity. Two variants (E215K and R440C) exhibited higher intrinsic clearance values than the wild-type protein, while the remaining 20 CYP2D6 allelic variants exhibited significantly decreased clearance values (2.01-87.56%) compared to CYP2D6*1. CONCLUSION: These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2D6 alleles when administering risperidone in the clinic. This is the first report of all these novel alleles for risperidone metabolism, providing fundamental data for further clinical studies on CYP2D6 alleles.

Inhibitory Effect of Apigenin on Pharmacokinetics of Venlafaxine in vivo and in vitro.

OBJECTIVE: This study was conducted to investigate the effects of orally administered apigenin on the pharmacokinetics of venlafaxine (VEN) in rats and on the metabolism of VEN in human and rat liver microsomes in vitro. METHODS: Ten healthy male SD rats were randomly divided into 2 groups: A group (control group), B group (a single dose of 250 mg/kg apigenin). A single dose of 20 mg/kg VEN was administered orally 30 min after administration of apigenin (250 mg/kg). VEN plasma levels were measured by HPLC with fluorescence detection, and pharmacokinetic parameters were calculated by DAS 3.0 software. RESULTS: The single dose of 250 mg/kg apigenin significantly increased the AUC0-t of VEN by 40.9% (p < 0.05) and obviously increased the peak plasma concentration (Cmax) of VEN (p < 0.05). Furthermore, apigenin showed inhibitory effect on human and rat microsomes and the IC50 of apigenin was 58.37 and 25.73 µmol/l, respectively. CONCLUSIONS: Our results indicated that an intake of apigenin could increase VEN plasma levels and some of its pharmacokinetic parameters (AUC, Tmax). Thus, more attention should be paid when VEN was administrated combined with apigenin.

Effects of cytochrome P450 2C9 polymorphism on bosentan metabolism.

Cytochrome P450 (P450) 2C9 is an important member of the P450 enzyme superfamily, with 58 CYP2C9 allelic variants previously reported. Genetic polymorphisms of CYP2C9 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to assess the catalytic activities of 38 human CYP2C9 alleles, including 24 novel alleles (*36-*60) found in the Han Chinese population, toward bosentan (BOS) in vitro. Insect microsomes expressing the 38 CYP2C9 alleles were incubated with 10-625 µM bosentan for 30 minutes at 37°C and terminated by cooling to -80°C immediately. BOS and hydroxyl bosentan, the major metabolite of BOS, were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry system. Thirty-eight defective alleles can be classified into three categories according to the relative clearance value compared with wild type: nine alleles exhibited significantly increased intrinsic clearance values (Vmax/Km) compared with the wild type (1.5-fold-∼4.9-fold relative clearance); nine alleles exhibited significantly reduced intrinsic clearance values compared with the wild type (0.6-28.9% relative clearance). The remaining 20 alleles exhibited no significant difference (1-fold) in enzyme activity compared with the wild type. These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2C9 alleles when administering BOS in the clinic. This is the first report of all these rare alleles for BOS metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles.

[Cigarette smoke extract reduces NOS activity and CX43 expression in the corporal cavernosum].

OBJECTIVE: To study the effects of different doses of cigarette smoke extract (CSE) on the erectile function of male rats and the mechanism of smoking-induced erectile dysfunction (ED). METHODS: A total of 75 healthy male SD rats were randomly divided into Groups A (control), B (dimethyl sulphoxide [DMSO]), C (low-dose CSE), D (medium-dose CSE) and E (high-dose CSE). CSE models were established in male SD rats by hypodermic injection, and 60 days later observed for penile erection following subcutaneous injection of apomorphine. Then the rats were killed and the penile cavernous body obtained for the examination of NOS activity by chromatometry and the determination of Cx43 expression by laser scanning confocal fluorescence microscopy (LCSM). RESULTS: Compared with the control and DMSO groups, penile erection frequency, NOS activity and Cx43 expression in the penile cavernous tissue were significantly decreased in the CSE groups (P < 0.05), and the decrease was proportional to the increase of the doses of CSE. No statistically significant differences were observed between the control and DMSO groups (P > 0.05). CONCLUSION: Cigarette smoke obviously reduces NOS activity and Cx43 expression in the penile cavernous tissue and seriously affects penile erection. The higher the dose, the more serious the influence. The decreases of NOS activity and Cx43 expression may be an important mechanism of ED.