Study on the mechanism of PM2.5 affecting Th1/Th2 immune imbalance through the notch signaling pathway in asthmatic mice.

Some research has shown that PM2.5 causes Th1/Th2 immune imbalance and aggravates asthma. However, the exact mechanism of PM2.5 causing aggravation of asthma remains unclear. The purpose of this study was to investigate whether exposure to PM2.5 exacerbates Th1/Th2 immune imbalance through the Notch signaling pathway. Eight-week-old SPF female BALF/c mice were sensitized by ovalbumin to establish an asthma mouse model. PM2.5 exposure was carried out by aerosol inhalation of PM2.5 (510 µg/m3) after each provocation. The lung function of mice was measured and Splenic T lymphocyte subsets were detected. Notch signaling pathway was tested. The levels of interferon (IFN)-γ and interleukin (IL)-4 in serum and bronchoalveolar lavage fluid were determined. The results showed that the expression of the mRNA and protein of Notch1 and Hes1 in the asthma group were significantly higher than those in healthy controls. The levels of IL-4 were also remarkably high; while the levels of IFN-γ were remarkably low in serum and BALF, the Th1% and Th1/Th2 ratios were significantly lower, and Th2% was significantly higher in the asthma group than in the healthy controls. PM2.5 promoted further activation of the Notch signaling pathway and aggravated Th1/Th2 immune imbalance in asthmatic mice. γ-secretase inhibitor can partially inhibit the activation of the Notch signaling pathway and alleviate aggravation of immune imbalance. In conclusion, the asthmatic mice had a Th1/Th2 immune imbalance and an overactivated Notch signaling pathway. PM2.5 further aggravated Th1/Th2 immune imbalance by activating the Notch signaling pathway.

Resveratrol attenuates cigarette smoke extract induced cellular senescence in human airway epithelial cells by regulating the miR-34a/SIRT1/NF-κB pathway.

Chronic obstructive pulmonary disease (COPD) is characterized by accelerated lung aging. Smoking is the critical risk factor for COPD. Cellular senescence of airway epithelial cells is the cytological basis of accelerated lung aging in COPD, and the regulation of microRNAs (miRNAs) is the central epigenetic mechanism of cellular senescence. Resveratrol (Res) is a polyphenol with anti-aging properties. This study investigated whether Res attenuates cigarette smoke extract (CSE)-induced cellular senescence in human airway epithelial cells (BEAS-2B) through the miR-34a/SIRT1/nuclear factor-kappaB (NF-κB) pathway. BEAS-2B cells were treated with Res, CSE and transfected with miR-34a-5p mimics. Cellular senescence was evaluated by senescence -related ß-galactosidase (SA-ß-gal) staining and expression of senescence-related genes (p16, p21, and p53). The expressions of miR-34a-5p, SIRT1, and NF-κB p65 were examined using quantitative real time polymerase chain reaction and western blotting. The senescence-associated secretory phenotype (SASP) cytokines (IL-1ß, IL-6, IL-8, TNF-α) were assessed by enzyme-linked immunosorbent assay. The binding between miR-34a-5p and SIRT1 was confirmed by dual-luciferase reporter assay. The results showed that CSE dose-dependently decreased cell viability and elevated cellular senescence, characterized by increased SA-ß-gal staining and senescence-related gene expressions (p16, p21, and p53). Further, CSE dose-dependently increased the expression of miR-34a-5p and SASP cytokines (IL-1ß, IL-6, IL-8, TNF-α) in BEAS-2B cells. Pretreatment with Res inhibited CSE-induced cellular senescence and secretion of SASP cytokines (IL-1ß, IL-6, IL-8, TNF-α) in a dose-dependent manner. Moreover, Res reversed the CSE-induced down-regulation of SIRT1 and up-regulation of miR-34a-5p and NF-κB p65. SIRT1 is a target of miR-34a-5p. Overexpression of miR-34a-5p via transfection with miR-34a-5p mimic in BEAS-2B cells attenuated the inhibitory effect of Res on cellular senescence, accompanied by reversing the expression of SIRT1 and NF-κB p65. In conclusion, Res attenuated CSE-induced cellular senescence in BEAS-2B cells by regulating the miR-34a/SIRT1/NF-κB pathway, which may provide a new approach for COPD treatment.

GABABR1 in DRN mediated GABA to regulate 5-HT expression in multiple brain regions in male rats with high and low aggressive behavior.

The identity of the mechanism that controls aggressive behavior in rodents is unclear. Serotonin (5-HT) and GABA are associated with aggressive behavior in rodents. However, the regulatory relationship between these chemicals in the different brain regions of rats has not been fully defined. This study aimed to clarify the role of GABABR1 in DRN-mediated GABA to regulate 5-HT expression in multiple brain regions in male rats with high and low aggressive behavior. Rat models of highly and less aggressive behavior were established through social isolation plus resident intruder. On this basis, GABA content in the DRN and 5-HT contents in the PFC, hypothalamus, hippocampus and DRN were detected using ELISA. Co-expression of 5-HT and GB1 in the DRN was detected by immunofluorescence and immunoelectron microscopy at the tissue and subcellular levels, respectively. GB1-specific agonist baclofen and GB1-specific inhibitor CGP35348 were injected into the DRN by stereotaxic injection. Changes in 5-HT levels in the PFC, hypothalamus and hippocampus were detected afterward. After modeling, rats with highly aggressive behavior exhibited higher aggressive behavior scores, shorter latencies of aggression, and higher total distances in the open field test than rats with less aggressive behavior. The contents of 5-HT in the PFC, hypothalamus and hippocampus of rats with high and low aggressive behavior (no difference between the two groups) were significantly decreased, but the change in GABA content in the DRN was the opposite. GB1 granules could be found on synaptic membranes containing 5-HT granules, which indicated that 5-HT neurons in the DRN co-expressed with GB1, which also occurred in double immunofluorescence results. At the same time, we found that the expression of GB1 in the DRN of rats with high and low aggressive behavior was significantly increased, and the expression of GB1 in the DRN of rats with low aggressive behavior was significantly higher than that in rats with high aggressive behavior. Nevertheless, the expression of 5-HT in DRN was opposite in these two groups. After microinjection of baclofen into the DRN, the 5-HT contents in the PFC, hypothalamus and hippocampus of rats in each group decreased significantly. In contrast, the 5-HT contents in the PFC, hypothalamus and hippocampus of rats in each group increased significantly after injection with CGP35348. The significant increase in GABA in the DRN combined with the significant increase in GB1 in the DRN further mediated the synaptic inhibition effect, which reduced the 5-HT level of 5-HT neurons in the DRN, resulting in a significant decrease in 5-HT levels in the PFC, hypothalamus and hippocampus. Therefore, GB1-mediated GABA regulation of 5-HT levels in the PFC, hypothalamus and hippocampus is one of the mechanisms of highly and less aggressive behavior originating in the DRN. The increased GB1 level in the DRN of LA-behavior rats exhibited a greater degree of change than in the HA-group rats, which indicated that differently decreased 5-HT levels in the DRN may be the internal mechanisms of high and low aggression behaviors.

Comparative studies of Toxoplasma gondii transcriptomes: insights into stage conversion based on gene expression profiling and alternative splicing.

BACKGROUND: Toxoplasma gondii is one of the most important apicomplexan parasites and infects one-third of the human population worldwide. Transformation between the tachyzoite and bradyzoite stages in the intermediate host is central to chronic infection and life-long risk. There have been some transcriptome studies on T. gondii; however, we are still early in our understanding of the kinds and levels of gene expression that occur during the conversion between stages. RESULTS: We used high-throughput RNA-sequencing data to assemble transcripts using genome-based and de novo strategies. The expression-level analysis of 6996 T. gondii genes showed that over half (3986) were significantly differentially expressed during stage conversion, whereas 2205 genes were upregulated, and 1778 genes were downregulated in tachyzoites compared with bradyzoites. Several important gene families were expressed at relatively high levels. Comprehensive functional annotation and gene ontology analysis revealed that stress response-related genes are important for survival of bradyzoites in immune-competent hosts. We compared Trinity-based de novo and genome-based strategies, and found that the de novo assembly strategy compensated for the defects of the genome-based strategy by filtering out several transcripts with low expression or those unannotated on the genome. We also found some inaccuracies in the ToxoDB gene models. In addition, our analysis revealed that alternative splicing can be differentially regulated in response to life-cycle change. In depth analysis revealed a 20-nt, AG-rich sequence, alternative splicing locus from alt_acceptor motif search in tachyzoite. CONCLUSION: This study represents the first large-scale effort to sequence the transcriptome of bradyzoites from T. gondii tissue cysts. Our data provide a comparative view of the tachyzoite and bradyzoite transcriptomes to allow a more complete dissection of all the molecular regulation mechanisms during stage conversions. A better understanding of the processes regulating stage conversion may guide targeted interventions to disrupt the transmission of T. gondii.

Effects of PM2.5 exposure on the Notch signaling pathway and immune imbalance in chronic obstructive pulmonary disease.

Chronic Obstructive Pulmonary Disease (COPD) is associated with T lymphocytes subset (Th1/Th2, Th17/Treg) imbalance. Notch signaling pathway plays a key role in the development of the adaptive immunity. The immune disorder induced by fine particulate matter (PM2.5) is related to COPD. The aim of this study was to investigate the mechanism by which PM2.5 influences the Notch signaling pathway leading to worsening immune disorder and accelerating COPD development. A COPD mouse model was established by cigarette smoke exposure. PM2.5 exposure was performed by aerosol inhalation. γ-secretase inhibitor (GSI) was given using intraperitoneal injection. Splenic T lymphocytes were purified using a density gradient centrifugation method. CD4+ T lymphocyte subsets (Th1/Th2, Th17/Treg) were detected using flow cytometry. mRNA and proteins of Notch1/2/3/4, Hes1/5, and Hey1 were detected using RT-PCR and Western blot. Serum INF-γ, IL-4, IL-17 and IL-10 concentrations were measured using ELISA. The results showed that in COPD mice Th1% and Th17%, Th1/Th2 and Th17/Treg were increased, and the levels of mRNA and protein in Notch1/2/3/4, Hes1/5, and Hey1 and serum INF-γ and IL-17 concentrations were significantly increased, and Th2%, Treg%, and serum IL-4 and IL-10 concentrations were significantly decreased. COPD Mice have Th1- and Th17-mediated immune disorder, and the Notch signaling pathway is in an overactivated state. PM2.5 promotes the overactivation of the Notch signaling pathway and aggravates the immune disorder of COPD. GSI can partially inhibit the activation of the Notch signaling pathway and alleviate the immune disorder under basal state and the immune disorder of COPD caused by PM2.5. This result suggests that PM2.5 is involved in the immune disorder of mice with COPD by affecting the Notch signaling pathway and that PM2.5 aggravates COPD.

Effects of Astragalus and Codonopsis pilosula polysaccharides on alveolar macrophage phagocytosis and inflammation in chronic obstructive pulmonary disease mice exposed to PM2.5.

Astragalus and Codonopsis pilosula are used for their immunomodulatory and anti-inflammatory effects. Here, we investigated the effects of Astragalus polysaccharides (APS) and Codonopsis pilosula polysaccharides (CPP) on alveolar macrophage (AM) phagocytosis and inflammation in chronic obstructive pulmonary disease (COPD) associated with exposure to particulate matter with a mean aerodynamic diameter ≤2.5µm (PM2.5). A mouse model of COPD was established by cigarette smoke exposure. PM2.5 exposure was performed by inhalation of a PM2.5 solution aerosol. APS and CPP were administered intragastrically. COPD showed defective AM phagocytosis and increased levels of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid and serum. PM2.5 exposure aggravated the damage, and this effect was reversed by APS and CPP gavage. The results indicate that APS and CPP may promote defective AM phagocytosis and ameliorate the inflammatory response in COPD with or without PM2.5 exposure.

Effects of resveratrol and genistein on nuclear factor­κB, tumor necrosis factor­α and matrix metalloproteinase­9 in patients with chronic obstructive pulmonary disease.

Chronic airway inflammation and airway remodeling are the major pathophysiological characteristics of chronic obstructive pulmonary disease (COPD). Resveratrol and genistein have been previously demonstrated to have anti­inflammatory and antioxidative properties. The present study aimed to measure the inhibitory effects of resveratrol and genistein on tumor necrosis factor (TNF)­α and matrix metalloproteinase (MMP)­9 concentration in patients with COPD. Lymphocytes were isolated from the blood of 34 patients with COPD and 30 healthy subjects, then randomly divided into the following four treatment groups: Control, dexamethasone (0.5 µmol/l), resveratrol (12.5 µmol/l) and genistein (25 µmol/l) groups. After 1 h of treatment, 100 µl lymphocytes were collected for nuclear factor (NF)­κB immunocytochemical staining. After 48 h treatment, the supernatant of the lymphocytes was collected for analysis of TNF­α and MMP­9 concentration levels. The percentage of lymphocytes with positive nuclear NF­κB expression was analyzed by immunocytochemical staining. The concentration levels of TNF­α and MMP­9 were measured using radioimmunoassay and enzyme­linked immunosorbent assay, respectively. The present study demonstrated that the percentage of NF­κB­positive cells, and the levels of TNF­α and MMP­9 in lymphocytes from patients with COPD patients were significantly higher compared with healthy subjects. Additionally, there were positive correlations between the percentage of NF­κB­positive cells, and the concentration levels of TNF­α and MMP­9 in patients with COPD. All three factors were significantly reduced in lymphocytes treated with resveratrol and genistein, and the inhibitory effects of resveratrol on NF­κB, TNF­α and MMP­9 were more potent than the effects of genistein. In conclusion, resveratrol and genistein may inhibit the NF­κB, TNF­α and MMP­9­associated pathways in patients with COPD. It is suggested that resveratrol and genistein may be potential drugs candidates for use in the treatment of COPD.

Sinomenine attenuates airway inflammation and remodeling in a mouse model of asthma.

Asthma is an inflammatory disease that involves airway inflammation and remodeling. Sinomenine (SIN) has been demonstrated to have immunosuppressive and anti­inflammatory properties. The aim of the present study was to investigate the inhibitory effects of SIN on airway inflammation and remodeling in an asthma mouse model and observe the effects of SIN on the transforming growth factor­ß1 (TGF­ß1)/connective tissue growth factor (CTGF) pathway and oxidative stress. Female BALB/c mice were sensitized by repetitive ovalbumin (OVA) challenge for 6 weeks in order to develop a mouse model of asthma. OVA­sensitized animals received SIN (25, 50 and 75 mg/kg) or dexamethasone (2 mg/kg). A blank control group received saline only. The area of smooth muscle and collagen, levels of mucus secretion and inflammatory cell infiltration were evaluated 24 h subsequent to the final OVA challenge. mRNA and protein levels of TGF­ß1 and CTGF were determined by reverse transcription­quantitative polymerase chain reaction and immunohistology, respectively. The indicators of oxidative stress were detected by spectrophotometry. SIN significantly reduced allergen­induced increases in smooth muscle thickness, mucous gland hypertrophy, goblet cell hyperplasia, collagen deposition and eosinophilic inflammation. The levels of TGF­ß1 and CTGF mRNA and protein were significantly reduced in the lungs of mice treated with SIN. Additionally, the total antioxidant capacity was increased in lungs following treatment with SIN. The malondialdehyde content and myeloperoxidase activities in the lungs from OVA­sensitized mice were significantly inhibited by SIN. In conclusion, SIN may inhibit airway inflammation and remodeling in asthma mouse models, and may have therapeutic efficacy in the treatment of asthma.

Dynamic oxidative stress and DNA damage induced by oestrogen deficiency and protective effects of puerarin and 17ß-oestradiol in ovariectomized rats.

Oxidative stress plays an essential role in the pathogenesis of cardiovascular diseases and osteoporosis resulting from oestrogen deficiency in the postmenopausal period. In this report, we observed a dynamic change of oxidative stress and DNA damage after ovariectomy in female rats. We then compared phytoestrogen puerarin and 17ß-oestradiol (E2) in their effects on oestrogen deficiency-induced oxidative stress and DNA damage. Serum total antioxidant capacity (TAC), malondialdehyde (MDA) and lymphocytes DNA damage (comet%) were measured. There was a gradual increase in oxidative stress in the ovariectomized (OVX) rats over time after ovariectomy, as compared to rats receiving sham operation. OVX rats that were on puerarin and E2 showed increased TAC and decreased MDA in the serum, as well as decreased lymphocytes comet%. Puerarin appeared to have a more powerful protective effect on DNA oxidative damage than E2. The study indicates that postmenopausal women may benefit from phytoestrogen puerarin.

[The relationship between coagulation/anticoagulation imbalance and oxidative stress in patients with chronic obstructive pulmonary disease].

OBJECTIVE: To explore the relationship between coagulation/anticoagulation imbalance and oxidative stress in the patients with chronic obstructive pulmonary disease during acute exacerbation (AECOPD) before and after treatment. METHODS: Plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) activity was detected by chromogenic assay in 28 AECOPD patients before and after treatment as well as in 30 healthy controls. The total antioxidative capacity (TAC), malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) in plasma were measured in both groups. RESULTS: The levels of plasma TF and TFPI, and their ratio (TF/TFPI) in AECOPD patients before treatment were significantly higher than those after treatment (all P < 0.01), the latter were still higher than those in the healthy persons (all P < 0.01). The levels of the TAC and GSH-PX in plasma in AECOPD patients before treatment were significantly lower than those after treatment (all P < 0.01), the latter were still lower than those in the healthy persons (all P < 0.01). The plasma MDA in AECOPD patients before treatment was significantly higher than that after treatment (P < 0.01), which was still higher than that in the healthy persons (P < 0.05). There were negative correlations between TF/TFPI ratio and TAC (r = -0.518, P < 0.01), GSH-PX (r = -0.454, P < 0.05), PaO2 (r = -0.511, P < 0.01) respectively and a positive correlation between TF/TFPI ratio and the percentage of neutrophils (r = 0.379, P < 0.05) in AECOPD patients before treatment. There still were negative correlations between TF/TFPI ratio and TAC (r = -0.420, P < 0.05), FEV(1)% to predicted (r = -0.480, P < 0.05) respectively, and a positive correlation between TF/TFPI ratio and MDA (r = 0.451, P < 0.05) in AECOPD patients after treatment. CONCLUSIONS: There existed coagulation/anticoagulation imbalance and oxidation/antioxidation imbalance before and after treatment in AECOPD patients and their relationship was explored.

[Effects of rosiglitazone on peroxisome proliferator activated receptor-γ pathway in patients with chronic obstructive pulmonary disease].

OBJECTIVE: To explore the effects of rosiglitazone on peroxisome proliferator activated receptor-γ (PPAR-γ), nuclear factor-κB and tumor necrosis factor-α (TNF-α) in patients with chronic obstructive pulmonary disease (COPD). METHODS: From Apr. 2010 to Nov. 2010, 30 patients with acute exacerbations of COPD, 22 males and 8 females, age 54 - 87 (mean 72 ± 9) years and 24 healthy controls, 18 males and 6 females, age 52 - 80 (mean 69 ± 10) years were included. The peripheral blood mononuclear cells (PBMCs) were isolated from venous blood and then cultured. On the basis of the treatment given, the PBMCs of COPD patients were divided into 3 groups: non-treatment group, rosiglitazone treatment group (rosiglitazone group) and rosiglitazone and GW9662 treatment group (combined treatment group). Cells from the healthy controls (control group) did not receive any drug treatment. The mRNA expression of PPAR-γ and NF-κB was measured with real-time PCR. The protein expression and nuclear translocation of PPAR-γ and NF-κB were detected using immunofluorescence with laser scanning confocal microscopy. The TNF-α level in culture supernatant was measured with ELISA. One-way ANOVA and LSD-t test and the Pearson correlation coefficient were used for statistical analysis. RESULTS: The mRNA and protein levels of PPAR-γ were lower in the non-treatment group (0.52 ± 0.10, 55 ± 11) than those in the control group (1, 85 ± 9), while the levels of NF-κB mRNA and protein were higher in the non-treatment group (1.69 ± 0.07, 145 ± 17) than those in the control group (1, 118 ± 7). The mRNA and protein levels of PPAR-γ in the rosiglitazone group (4.47 ± 0.11, 204 ± 12) were significantly increased compared with the non-treatment group, while the mRNA and protein levels of NF-κB (0.33 ± 0.04, 59 ± 14) were remarkably decreased compared with the non-treatment group. The mRNA and protein levels of PPAR-γ (2.25 ± 0.31, 142 ± 23) were significantly decreased in the combined treatment group compared to the rosiglitazone group, but higher compared with the non-treatment group, while the mRNA and protein levels of NF-κB (0.64 ± 0.02, 90 ± 10) were increased compared with the rosiglitazone group, but decreased compared to the non-treatment group (F = 29.21 - 567.42, all P < 0.01). The TNF-α level was significantly higher in the non-treatment group (96.2 ± 1.4) µg/L than that in the control group (85.3 ± 1.0) µg/L. The TNF-α level in the rosiglitazone group (63.0 ± 2.5) µg/L was remarkably decreased compared with the non-treatment group, while that in the combined treatment group (83.3 ± 1.9) µg/L was increased compared with the rosiglitazone group, but decreased compared to the non-treatment group (F = 293.72, P < 0.01). The proteins of PPAR-γ and NF-κB were respectively located in cytoplasm and in nucleus in the non-treatment group, meanwhile they were located in both cytoplasm and nucleus in the control group. PPAR-γ protein was translocated from cytoplasm into nucleus and NF-κB protein was translocated from nucleus into cytoplasm in the rosiglitazone group. In the combined treatment group, PPAR-γ protein translocated from nucleus into cytoplasm and NF-κB protein partly translocated from cytoplasm into nucleus. By linear correlation analysis, PPAR-γ protein was negatively correlated with NF-κB protein and TNF-α level (r = -0.935, -0.924, all P < 0.01), while NF-κB protein was positively correlated to TNF-α level (r = 0.846, P < 0.01). CONCLUSIONS: The expression and activity of PPAR-γ were decreased in COPD patients. PPAR-γ agonist rosiglitazone inhibited inflammation in COPD through upregulating the expression and activity of PPAR-γ and inhibition of NF-κB and TNF-α. It suggests that PPAR-γ may play an important role in the inflammation of COPD.

The effects of genistein and puerarin on the activation of nuclear factor-kappaB and the production of tumor necrosis factor-alpha in asthma patients.

Oxidative stress might play an essential role in the pathogenesis of chronic airway inflammatory diseases, indicating that antioxidant therapy may have a potential effect in controlling chronic airway inflammatory diseases. The aim of the present study was to investigate the effect of antioxidants genistein and puerarin on the activation of nuclear factor-kappaB (NF-kappaB) and the production of tumor necrosis factor-alpha (TNF-alpha) in peripheral blood mononuclear cells (PBMCs) in asthma patients. PBMCs were isolated from blood samples of 32 asthma patients and 31 healthy persons, and randomly divided into four groups, control group, dexamethasone group, genistein group and puerarin group. The expression of NF-kappaB in nuclei was analysed by immunocytochemical staining. The level of TNF-alpha was measured by radioimmunoassay. Results showed that the percentage of NF-kappaB positive cells in PBMCs and the level of TNF-alpha in PBMCs supernatants were significantly higher in asthma patients 23.1 +/- 6.7%, 2.10 +/- 0.38 microg/L than in those of healthy persons 7.2 +/- 2.9%, 0.86 +/- 0.53 microg/L (p all < 0.01). There was a positive correlation between the percentage of NF-kappaB positive cells and the level of TNF-alpha in asthma patients (r = 0.709, p < 0.01). The percentages of NF-kappaB positive cells in PBMCs were significantly decreased in the genistein group 15.2 +/- 5.4% and in the puerarin group 16.2 +/- 5.1% than those in control groups 23.1 +/- 6.7% in asthma patients (p respectively < 0.01, < 0.05). The levels of TNF-alpha in PBMCs supernatants were remarkably decreased in genistein group 1.08 +/- 0.40 microg/L and in puerarin group 1.24 +/- 0.29 microg/L than those in control group 2.10 +/- 0.38 microg/L in asthma patients (p all < 0.01). There were positive correlations between the percentages of NF-kappaB positive cells and the levels of TNF-alpha in genistein group (r = 0.579, p < 0.01) and in puerarin group (r = 0.665, p < 0.01) in asthma patients. These results show that the activation of NF-kappaB and TNF-alpha pathway of PBMCs may play an important role in the pathogenesis of asthma. Genistein, and puerarin could inhibit the pathway of NF-kappaB and TNF-alpha in asthma patients, so it was implicated that asthma patients will benefit from the antioxidants genistein and puerarin.

[Effects of matrine on airway inflammation and early airway remodeling in asthmatic mice].

OBJECTIVE: To observe the influence of matrine on airway inflammation and early airway remodeling in asthmatic mice. METHODS: Fifty BALB/c mice were randomly divided into 5 groups: a normal control group (A), an asthmatic group (B), a dexamethasone (DXM) group (C, 2 mg/kg), a high-dose matrine group (D, 50 mg/kg) and a low-dose matrine group (E, 25 mg/kg). The mice model of asthma in the B, C, D, and E groups was established by ovalbumin (OVA) intraperitoneal injections and aerosolization. Intra-gastric administration of different medications in C, D, E groups and 0.9% sodium chloride in B group were carried out 1 hour before provocation. 0.9% sodium chloride was used for intraperitoneal injection, aerosolization and intra-gastric administration in group A. The lung tissue slices were stained, and then the grade of inflammation around the wall of bronchi, mucous secretion, and the percentage of goblet-cells were counted. The areas of bronchial smooth muscle and of collagen deposition in airway wall were analyzed. The transcriptions and protein expressions of transforming growth factor-beta(1) (TGF-beta(1)) and connective tissue growth factor (CTGF) were measured respectively by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: In the A, B, C, D, E groups, the grades of inflammation were 1.5 (1, 2), 4 (4, 5), 2 (1, 3), 2 (2, 3), 3 (2, 3.3), respectively; the degrees of mucous secretions were 1.5 (1, 2), 5 (4, 6), 2 (1, 3), 2 (2.5, 4), 3 (3, 4), respectively. These airway inflammatory parameters in group B were significantly higher than in group A (chi(2) = 21.3, 22.6, P all < 0.01), while they were remarkably decreased in group C compared to group B (chi(2) = 13.3, 15.0, P all < 0.01). These parameters in group D and group E were also lower than those in group B (chi(2) = 9.1, 10.9; 9.8, 9.7; P all < 0.05). The percentage of goblet cells in airway epithelium was (1.7 +/- 0.5)%, (54.7 +/- 15.5)%, (20.4 +/- 5.9)%, (31.7 +/- 7.6)% and (36.2 +/- 10.8)%, respectively; it was significantly higher in group B than in group A (t = 12.0, P < 0.01), and remarkably lower in groups C and D than in group B (t = 7.7, 5.1, P all < 0.01), and lower in group E than in B group (t = 4.2, P < 0.05). In these 5 groups, the area of bronchial smooth muscle was (11.5 +/- 2.1) microm(2)/microm, (30.0 +/- 3.3) microm(2)/microm, (15.2 +/- 3.1) microm(2)/microm, (22.2 +/- 4.8) microm(2)/microm and (26.5 +/- 3.4) microm(2)/microm, respectively; it was significantly higher in group B than in group A (t = 11.4, P < 0.01), and remarkably lower in groups C, D and E than in group B (t = 9.1, 4.7, 2.2, P all < 0.01). The area of collagen deposition was (3.9 +/- 1.8) microm(2)/microm, (24.4 +/- 6.1) microm(2)/microm, (15.4 +/- 3.5) microm(2)/microm, (16.6 +/- 6.0) microm(2)/microm and (17.5 +/- 4.4) microm(2)/microm, respectively; it was also significantly higher in group B than in group A (t = 9.3, P < 0.01), and remarkably lower in groups C and D than in group B (t = 4.1, 3.5, P all < 0.01), and lower in group E than in B group (t = 3.2, P < 0.05). The mRNA levels of TGF-beta(1) were 160 +/- 25, 247 +/- 37, 174 +/- 23, 195 +/- 25 and 207 +/- 42, respectively, and those of CTGF were 86 +/- 8, 160 +/- 24, 94 +/- 10, 93 +/- 14 and 104 +/- 10, respectively in the 5 groups. The levels were remarkably increased in group B, as compared to group A (t = 6.1, 11.6, P all < 0.01), and the levels in groups C, D and E were remarkably decreased, as compared to group B, the difference being significant (t = 3.7, 2.7, 5.1; 10.6, 8.6, 10.3; P all < 0.01). The protein level of TGF-beta(1) in lung tissues was 21 +/- 5, 36 +/- 8, 26 +/- 5, 26 +/- 5 and 26 +/- 5, respectively, and that of CTGF was 15 +/- 4, 27 +/- 5, 21 +/- 4, 22 +/- 3 and 23 +/- 4, respectively in the 5 groups. The levels in B group were significantly increased, as compared to group A (t = 5.7, 6.4, P all < 0.01), and those in groups C and D were significantly decreased (t = 3.9, 3.9; 3.2, 2.8, P all < 0.01), and that in group E was also lower (t = 3.8, 2.5, P all < 0.05), as compared to group B. In all the groups, the protein levels of TGF-beta(1) and CTGF were positively correlated with the area of bronchial smooth muscle and with the area of collagen deposition (r = 0.435, 0.583, 0.522, 0.590, P all < 0.01). CONCLUSIONS: Matrine inhibited airway inflammation and early airway remodeling in asthmatic mice. The signal transduction of TGF-beta(1) and CTGF maybe involved.

Antioxidative and immunomodulatory role of melatonin, sodium selenite, N-acetyl-L-cysteine and quercetin on human umbilical blood.

We have previously reported on the DNA oxidative damage and oxygen stress in healthy term neonates. The aim of the present study was to investigate the antioxidative and immunomodulatory role of melatonin, sodium selenite, N-acetyl-L-cysteine and quercetin on umbilical blood. The single cell gel electrophoresis (comet) assay was used for DNA oxidative damage. The lymphocytes proliferation and natural killer (NK) activity in umbilical blood were assayed by 3[H]-thymidine uptaken and MTT methods. Results using comet assay showed protection by melatonin, N-acetyl-L-cysteine (NAC) and quercetin, and a protective and damaging effect by selenite sodium. Melatonin, sodium selenite, NAC and quercetin greatly promoted the lymphocytes proliferation to IL-2. NK activity of the umbilical blood was significantly increased by melatonin and sodium selenite, but was not affected by NAC and quercetin. These data suggest that antioxidants are able to protect umbilical blood mononuclear cells against oxygen stress and affect oxygen stress mediated immune function inhibition of umbilical blood. These results will supply new experimental data for using umbilical blood to treat diseases.

Oxidative stress in Graves' disease patients and antioxidant protection against lymphocytes DNA damage in vitro.

DNA damage to peripheral blood lymphocytes of patients with Graves' disease (GD) was studied in vitro before and after treatment with antioxidants, melatonin, quercetin, N-acetylcysteine (NAC) and vitamin C. DNA damage (comet %) was remarkably higher in patients (23.7 +/- 5.5%) than that in healthy persons (9.8 +/- 3.2%, p < 0.01). Plasma malondialdehyde (MDA) content (7.90 +/- 1.77 microM) of patients was significantly higher than that of healthy persons (4.71 +/- 1.19 microM, p < 0.01). Also, the plasma total antioxidant capacity (TAC) (7.53 +/- 1.35 U/ml) in GD patients was significantly lower than that in healthy persons (10.56 +/- 2.21 U/ml, p < 0.01). Negative correlations were observed between plasma TAC and DNA damage in lymphocytes (r = -0.599, p < 0.01), and between plasma TAC and MDA (r = -0.40, p < 0.05) in GD patients. After treatment with 100 microM melatonin, quercetin or NAC for 4 h in vitro, DNA damage in lymphocytes in GD patients declined significantly (from 23.8 +/- 4.4% to 14.4 +/- 4.0%, p < 0.001 for melatonin, from 23.4 +/- 4.7% to 18.1 +/- 4.3%, p < 0.01 for quercetin, from 23.7 +/- 4.0% to 18.7 +/- 5.7%, p < 0.05 for NAC), while there was little change with concentrations of 1-100 microM of vitamin C. However, 1000 microM vitamin C enhanced DNA damage significantly (from 23.8 +/- 2.3% to 30.3 +/- 3.9%, p < 0.05). Our results showed that oxidative stress existed in GD patients and the antioxidants melatonin, quercetin and NAC are beneficial for DNA damage in lymphocytes of GD patients in vitro.

DNA damage in healthy term neonate.

BACKGROUND: The process of childbirth is accompanied by an increase in oxidative aggression. AIM: To determine DNA damage and oxidative stress in healthy term neonates at birth. DESIGN: A total of 34 healthy full-term neonates, 22 healthy adults and 20 samples of colostrum from mothers of full-term neonates were examined. The malondialdehyde (MDA), DNA damage, GSH/GSSG ratio and total antioxidant capacity (TAC) in mononuclear cells isolated from umbilical blood and adult peripheral blood were measured. Moreover, the TAC of colostrum was also measured. The protective activity of five natural polyphenols against H(2)O(2)-induced DNA damage in mononuclear cells of umbilical blood was studied. RESULTS: A high level of DNA damage (p<0.001) accompanied with lower TAC (p<0.05) and GSH/GSSG ratio (p<0.001) and with higher level of MDA (p<0.001) in umbilical blood compared with those of healthy adult peripheral blood. The natural polyphenols, 7,8-dihydroxy-4-methyl coumarin, quercetin and resveratrol, are able to protect mononuclear cells of umbilical blood from oxidative attack. However, other two polyphenols, rutin and 7-hydroxy-4-methyl coumarin, do not. The TAC of colostrum is significantly higher than that of umbilical blood (p<0.001). CONCLUSIONS: The DNA oxidative damage in mononuclear cells of umbilical blood as well as other indexes related to redox status provided evidence that a sudden increase in oxygenation exposes the neonate into oxidative stress. Colostrum with a significant high TAC is very important for health care in infants against the oxidative stress.

[A study on oxidative DNA damage and lipid peroxidation in patients with tuberculous pleurisy].

OBJECTIVE: To explore the levels of mononuclear cell oxidative DNA damage and lipid peroxidation in patients with tuberculous pleurisy. METHODS: The mononuclear cell DNA damages in pleural effusion and peripheral blood of 28 patients with tuberculous pleurisy and in peripheral blood of 25 healthy persons were detected by single cell gel electrophoresis (comet%). The levels of total antioxidative capacity (TAC) in supernatant of pleural effusion and blood plasma of 28 patients and in blood plasma of 25 healthy persons were measured by o-phenanthroline colorimetric analysis. The contents of malondialdehyde (MDA) of blood plasma from 28 patients and from 25 healthy persons were measured by thiobarbituric acid colorimetric analysis. The differences between groups were analyzed by t test. RESULTS: The comet percentage of mononuclear cells in pleural effusion from patients with tuberculous pleurisy was (41.3 +/- 14.5)%, which was significantly higher than that in the peripheral blood (21.2 +/- 4.2)% (P < 0.01); The level of TAC in supernatant of pleural effusion was (5 172 +/- 1 195) U/L, which was lower than that in the blood plasma (8 656 +/- 1 592) U/L (P < 0.01). There was a negative correlation between the comet percentage of mononuclear cells and the level of TAC (r = -0.425, P < 0.05) in pleural effusion. The comet percentage of mononuclear cells in peripheral blood and the content of MDA in blood plasma from patients were (21.2 +/- 4.2)% and (8.25 +/- 1.37) micro mol/L, which were significantly higher than those from the controls (8.9 +/- 3.7)% and (4.46 +/- 0.93) micro mol/L respectively (P < 0.01); The level of TAC in blood plasma was (8 656 +/- 1 592) U/L, which was lower than that from the controls (10 610 +/- 1 399) U/L (P < 0.01). There were negative correlations between the level of TAC in blood plasma and the comet percentage of mononuclear cells in peripheral blood or the content of MDA in blood plasma (r = -0.438, -0.413, P < 0.05), and there was a positive correlation between the comet percentage of mononuclear cells in peripheral blood and the content of MDA in blood plasma (r = 0.899, P < 0.01). CONCLUSION: There is oxidation/antioxidation imbalance in patients with tuberculous pleurisy, namely oxidative stress, with inadequate total antioxidative capacity, particularly in the diseased pleural cavity.