Effects of Bone Marrow-derived Mesenchymal Stem Cells on TGF-β and MCP-1 in Injured Lung of Rats

The primary objective of the present study is to investigate the therapeutic effect of bone marrow-derived mesenchymal stem cells （MSCs） on bleomycin （BLM）-induced lung injury of rats and the effect on transforming growth factor-β （TGF-β） and monocyte chemoattractant protein-1 （MCP-1）. MSCs were isolated from SD rats. The recipients rats were divided randomly into four groups： lung injury group, MSC treatment group, MSC control group and normal control group. Rats of lung injury group and MSC treatment group were perfused with BLM of 5mg/kg （0.2-0.3ml） intratracheally, others were perfused with normal saline. After twelve hours, rats of MSC treatment group and MSC control group were injected MSCs of 0.5×10^6per rat into tail vein. Haematoxylin-eosin staining was used to observe the morphology in lung tissue. ELISA was used to detect the contents of TGF-β and MCP-1 in serum and bronchoalveolar lavage fluid （BALF）. Collagen content of the lung tissue was assessed by hydroxyproline （HYP） concentration. It was found that the thickness of alveolar wall and lung interstitium were significantly reduced in the rats of MSC treatment group compared with the lung injury group. HYP content in lung interstitium, TGF-β and MCP-1 in serum and BALF were increased significantly in rats of lung injury group two weeks after BLM perfusion, but they were reduced significantly in the rats of MSC treatment group compared with the injured rats. These observations provide evidence that MSCs engraftment could alleviate bleomycin-induced lung injury and fibrosis in rats and the therapeutic effects might relate with the decrease of TGF-β and MCP-1.     

Changes of Protein Kinase Cα and Cyclin D1 Expressions in Pulmonary Arteries from Smokers with and without Chronic Obstructive Pulmonary Disease

The purpose of this study was to investigate the changes of protein kinase Ca （PKCα） and cyclin D1 expressions in pulmonary arteries from＇smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease （COPD）. The peripheral lung tissues were obtained from 10 non-smokers with normal lung function （non-smoker group）, 14 smokers with normal lung function （smoker group）, 11 smokers with mild to moderate COPD （COPD group）. The morphological changes of pulmonary arteries were observed by HE-staining. The expressions of ct-smooth muscle actin （α-SMA）, proliferating cell nuclear antigen （PCNA）, PKCα and cyclin D1 proteins in pulmonary artery smooth muscle cells （PASMCs） were immunohistochemically determined. The percentages of PCNA-positive cells were taken as the smooth muscle cells proliferation index （PI）. The mRNA expressions of PKCα and cyclin D l in PASMCs were evaluated by real-time fluorescence PCR. Morphometrical analysis showed that the ratio of pulmonary artery wall area to total area （WA%） in smoker group and COPD group was significantly greater than that in non-smoker group （P〈0.01）. The PASMCs proliferation index in smoker group and COPD group was significantly higher than that in nonsmoker group （P〈0.01）. The protein levels of PKCct and cyclin D1 inPASMCs were significantly increased in smoker group and COPD group as compared with non-smoker group （P〈0.01）. The mRNA expressions of PKCα and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with non-smoker group （P〈0.01）. Significant correlations were found between PKCα protein and WA% or PI （P〈0.01）. Correlations between cyclin D1 protein and WA% or PI also existed （P〈0.01）. The expression of PKCa was positively correlated with the expression of cyclin D 1 at both protein and mRNA levels （P〈0.01）. In conclusion, increased expressions of PKCα and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.     

Inflammatory airway features and hypothalamic-pituitary- adrenal axis function in asthmatic rats combined with chronic obstructive pulmonary disease

Background Bronchial asthma （BA） and chronic obstructive pulmonary disease （COPD） are both inflammatory airway diseases with different characteristics. However, there are many patients who suffer from both BA and COPD. This study was to evaluate changes of inflammatory airway features and hypothalamic-pituitary-adrenal （HPA） axis function in asthmatic rats combined with COPD. Methods Brown Norway （BN） rats were used to model These three models were compared and evaluated with the inflammatory airway diseases of BA, COPD and COPD＋BA. respect to clinical symptoms, pulmonary histopathology, airway hyperresponsiveness （AHR）, inflammatory cytokines and HPA axis function. Results The inflammatory airway features and HPA axis function in rats in the COPD＋BA model group were greatly influenced. Rats in this model group showed features of the inflammatory diseases BA and COPD. The expression of inflammatory cytokines in this model group might be up or downregulated when both disease processes are present. The levels of corticotrophin releasing hormone mRNA and corticosterone in this model group were both significantly decreased than those in the control group （P 〈0.05）. Conclusions BN rat can be used as an animal model of COPD＋BA. By evaluating this animal model we found that the features of inflammation in rats in this model group seem to be exaggerated. The HPA axis functions in rats in this model group have been disturbed or impaired, which is prominent at the hypothalamic level.     

Expression of nerve growth factor and hypoxia inducible factor-1α and its correlation with angiogenesis in non-small cell lung cancer

Summary： In order to investigate the expression of nerve growth factor （NGF） and hypoxia inducible factor-1α （HIF-1α） and its correlation with angiogenesis in non-small cell lung cancer （NSCLC）, paraffin-embedded tissue blocks from 20 patients with NSCLC were examined. Twenty corresponding para-cancerous lung tissue specimens were obtained to serve as a control. The expression of NGF, HIF-1α, and vascular endothelial growth factor （VEGF） in the NSCLC tissues was detected by using immunohistochemistry. The microvascular density （MVD） was determined by CD31 staining. The resuits showed that the expression levels ofNGF, HIF-1α and VEGF in the NSCLC tissues were remarkably higher than those in the para-cancerous lung tissues （P〈0.05）. There was significant difference in the MVD between the NSCLC tissues （9.19±1.43） and para-cancerous lung tissues （2.23±1.19） （P〈0.05）. There were positive correlations between NGF and VEGF, between HIF-1α and VEGF, and between NGF and HIF-1α in NSCLC tissues, with the spearman correlation coefficient being 0.588, 0.519 and 0.588, respectively. In NSCLC tissues, the MVD had a positive correlation with the three factors （P〈0.05）. Theses results suggest that NGF and HIF-1α are synergically involved in the angiogenesis of NSCLC.     

Pathogenesis of cigarette smoke-induced chronic obstructive pulmonary disease and therapeutic effects of glucocorticoids and N-acetylcysteine in rats

Background T lymphocytes and matrix metalloproteinase (MMP) play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the details of the mechanisms involved are unclear. The aims of this study were to investigate the changes in interferon-γ (IFN-γ), interleukin-4 (IL-4), MMP-9, MMP-12 and tissue inhibitor of metalloproteinase-1 (TIMP-1 ) levels in a smoke-induced COPD rat model and the therapeutic effects of glucocorticoids and Nacetylcysteine.Methods Male Wistar rats were exposed to cigarette smoke for 3.5 months. Budesonide or Nacetylcysteine was given in the last month. Lung function was measured at the end of the study. IL-4 and IFN-γ levels were then determined in bronchoalveolar lavage fluid and lung tissue samples by enzyme-linked immunosorbent assay. The expression of MMP-9, MMP-12 and TIMP-1 mRNA in lung tissue was determined by RT-PCR.Results In comparison with the control group, rats exposed to smoke had a significant increase in IL-4 and MMP-12 levels and a significant decrease in IFN-γ levels. In addition, the IL-4/ IFN-γ ratio and MMP-12/TIMP-1 ratio were both higher. At the same time, the ratio of forced expiratory volume in 0.3 second to forced vital capacity (FEV0.3/FVC) and dynamic compliance (Cdyn) decreased and expiratory resistance (Re) increased. By measuring pulmonary mean linear intercept and mean alveolar numbers, obvious emphysematous changes were observed in the smoke exposed group.After treatment with budesonide, IL-4 and MMP-12 decreased and IFN-γ increased. The IL-4/IFN-γ ratio returned to normal, though the MMP-12/TIMP-1 ratio remained unchanged. FEV0 JFVC was significantly higher and Re was significantly lower than that in untreated smoke exposed rats. No significant differences were found in pulmonary mean linear intercept and mean alveolar numbers.After treatment with N-acetylcysteine, IFN-γ increased and the IL-4/IFN-y ratio decreased. The MMP-12/TIMP-1 ratio remained unchanged. Re and Cdyn both improved obviously. No significant differences were found in pulmonary mean linear intercept and mean alveolar numbers. Correlation analysis indicated that IL-4 levels in lung tissue correlated negatively with FEV0.3/FVC (r=-0.53, P=0.001 ), IFN-γ levels in lung tissue correlated negatively with Re ( r=-0.63, P=0. 000) andpositively with Cdyn ( r=0.44, P=0. 009), and that the IL-4/IFN-γ ratio correlated negatively with FEV0.3/FVC ( r=-0.44, P=0.010 ) and Cdyn ( r=-0.42, P=0. 015 ) and positively with Re ( r=0.58, P=0.000). Finally, MMP-12 correlated negatively with FEV0.3/FVC (r=-0.36, P=0.026).Conclusions Cigarette smoke exposure increases IL-4 levels and decreases IFN-γ levels. This may be the result of smoke-induced changes in lung function. Budesonide can mitigate the chanqes in IL-4 and IFN-γ levels induced by smoke exposure.N-acetylcysteine has no effect on IL-4, but increases IFN-γ levels and brings the IL4/ IFN-y ratio back to normal. Cigarette smoke can also promote MMP-12 gene expression and elevate the MMP-12/TIMP-1 ratio. This effect may play a role in smokeinduced emphysema. Budesonide and Nacetylcysteine do not alter the MMP-12/TIMP-1 ratio in this study when given in the late phase of smoke exposure.     

CD8+ Tc-lymphocytes immunodeviation in peripheral blood and airway from patients of chronic obstructive pulmonary disease and changes after short-term smoking cessation

Background Cigarette smoke induces an acute but persisting inflammation in peripheral blood and airway in chronic obstructive pulmonary disease （COPD）,and CD8＋ Tc-lymphocytes are considered as a key role in this process.We aimed to investigate the Tc-lymphocytes immunodeviation in system and local airway of COPD patients and changes of the immunodeviation after short-term smoking cessation.Methods Peripheral blood （PB） and bronchoalveolar lavage fluid （BALF） were collected from 42 patients （14 COPD patients,16 smokers with normal lung function and 12 nonsmokers）,while PB and induced sputum （IS） were obtained from other 19 patients （10 quitting smokers and 9 continuing smokers） at baseline and follow-up respectively of 4-week smoking cessation.Percentages of CD8＋ Tc-lymphocytes （%CD3＋） and Tc1/Tc2 ratios were measured by flow cytometry.Results Percentages of CD8＋ Tc-lymphocytes were higher in COPD patients than those in smokers and nonsmokers in both PB and BALF.Tc1/Tc2 ratio in PB and in BALF from COPD patients was greater than that from smokers and nonsmokers and negatively correlated with FEV1%pre.When comparing the ratios between PB and BALF,significantly positive correlation was found in COPD patients.Furthermore,after 4-week smoking cessation,percentages of CD8＋ Tc-lymphocytes in PB and IS in quitting smokers were decreased compared to that in baseline and continuing smokers,whereas Tc1/Tc2 ratios were not influenced.Conclusions CD8＋ Tc1-trend immunodeviation profiles occurred in both system and local airway of COPD patients.This exceptional immunodeviation could not be relieved by short-term smoking cessation.     

Protective Effect of Hydroxysafflor Yellow A on Inflammatory Injury in Chronic Obstructive Pulmonary Disease Rats

Objective: To investigate the attenuating effect of Hydroxysafflor yellow A(HSYA) on inflammatory injury in chronic obstructive pulmonary disease(COPD). Methods: Rats were randomly assigned to 7 groups according to body weight including normal control group, HSYA blank group(76.8 mg/kg), COPD group, COPD+HSYA(30, 48, 76.8 mg/kg) groups and COPD+dexamethasone(2 mg/kg), 10 in each group. Passive cigarette smoke and intratracheal instil ation of lipopolysaccharides were used to establish a COPD model in rats. Hematoxylin and eosin staining of lung tissue sections was used, real-time polymerase chain reaction(PCR) was used to assay m RNA levels of some cytokines in lung tissues, the cytokines in bronchoalveolar lavage fluid(BALF) were measured by enzyme-linked immunosorbent assay(ELISA), Western blot analysis was used to determine phosphorylated p38 mitogen-activated protein kinase(MAPK) levels in lung tissues, and nuclear factor-κB(NF-κB) p65 protein levels in lung tissues were detected by immunohistochemistry. Results: Lung alveolar septa destruction, alveolus fusion, inflammatory cel infiltration, and bronchiole exudation were observed. These pathological changes were al eviated in the COPD+HSYA group. The m RNA expression of inflammatory factors were significantly increased in lung tissues from COPD rats(all P0.01) and were inhibited by HSYA. Levels of inflammatory cytokines in BALF of COPD rats were significantly increased(all P0.01) which were inhibited by HSYA(all P0.01, 48, 76.8 mg/kg). The levels of p38 MAPK phosphorylation and p65 in lung tissues of COPD rats were significantly increased(al P0.01) and were suppressed by HSYA(all P0.01, 48, 76.8 mg/kg). Conclusions: HSYA could alleviate inflammatory cell infiltration and other pathological changes in the lungs of COPD rats. HSYA could inhibit inflammatory cytokine expression, and increase phosphorylation of p38 MAPK and NF-κB p65 in the lungs of COPD rats. The protective mechanism of HSYA to inhibit COPD inflammation might be by attenuating NF-κB and p38 MAPK signal transduction.     

Inhibition of tumor necrosis factor-α reduces alveolar septal cell apoptosis in passive smoking rats

Background Recent studies have revealed that lung cell apoptosis plays an important role in pathogenesis of cigarette-induced chronic obstructive pulmonary disease （COPD）. Tumor necrosis factor alpha （TNF-α） is one of the most important cytokines which are involved in COPD. This study aimed at investigating the influence of its inhibitor, recombinant human necrosis factor-alpha receptor II：lgG Fc fusion protein （rhTNFR：Fc） on alveolar septal cell apoptosis in passive smoking rats. Methods Forty-eight rats were randomly divided into a normal control group, a passive smoking group, an rhTNFR：Fc intervention group and a sham intervention group. The passive smoking rats were treated by exposure to cigarette smoking daily for 80 days. After smoking for one month the rhTNFR：Fc intervention group was treated with rhTNFR：Fc by subcutaneous injection, the sham intervention group injected subcutaneously with a neutral preparation （normal saline 0.1 ml, manicol 0.8 ml, cane sugar 0.2 mg, Tris 0.024 mg as a control. Lung function was determined and the levels of TNF-a in serum and broncho-alveolar lavage fluid （BALF） were measured with enzyme-linked immunosorbnent assay （ELISA）. Lung tissue sections stained by hematoxylin and eosin （HE） were observed for study of morphological alternations. Mean linear intercept （MLI） and mean alveolar numbers （MAN） were measured and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） method was carried out to determine the percentage of positive cells and distribution of apoptotic cells. Results Increased MLI and decreased MAN were found in the passive smoking group compared with both the normal control group and the rhTNFR：Fc intervention group （P〈0.05）. Forced expiratory volume in 0.3 second （FEVo.3）/forced vital capacity （FVC） and peak expiratory flow （PEF） were lower in the passive smoking group than that in the normal control group （P〈0.05）. Compared with the sham intervention group     

Effect of valsartan on the expression of angiotensin II receptors in the lung of chronic antigen exposure rats

Background Many studies have suggested that angiotensin Ⅱ （Ang Ⅱ） and its receptors may be involved in the development of asthma. However, the expression of angiotensin Ⅱ receptors （AGTR） is not clear in the lung tissue of chronic asthmatics. This study was designed to determine the relationship between airway remodeling, dysfunction and the expression of AGTRs in a rat model of asthma. Methods Rats were sensitized with ovalbumin （OVA） for 2 weeks. Sixty minutes before an inhalation challenge, the rats were pretreated either with valsartan （15, 30, 50 mg.kg-1.d-1） or saline intragastrically. Then the rats received an OVA challenge for 30 alternative days. Acetylcholine （Ach）-induced bronchoconstriction was measured after the final antigen challenge. White cell counts in bronchoalveolar lavage fluid （BALF） and morphological changes in the airways were then assessed. The levels of transforming growth factor-beta 1 （TGF-β1） and platelet-derived growth factor （PDGF） in BALF were detected by ELISA. The levels of AGTR1 and AGTR2 mRNA and protein in lung tissues were measured by RT-PCR and Western blotting. Results AGTR1 mRNA and protein levels in repeatedly OVA-challenged rats were significantly increased as compared with negative controls. The AGTR1 mRNA expression versus white cell counts of BALF and airway wall thickness （mainly in small airways） in lungs of chronic antigen-exposed rats were positively correlated. Valsartan decreased the level of AGTR1 in repeatedly OVA-challenged rats. However, AGTR2 mRNA and protein levels in the OVA-challenged rats and high-dose valsartan-treated rats （50 mg.kg-1.d-1） were also increased. Valsartan significantly decreased inflammatory cell accumulation and attenuated Ach-evoked bronchoconstriction in repeatedly antigen-challenged rats. Valsartan also decreased allergen-induced structural changes in rat airway （including total airway wall thickness and smooth muscle area） and the levels of TGF-β1 and PDGF in BALE Conclusions AGTR     

Immunological effect of PM2.5 on cytokine production in female Wistar rats

Objective To investigate the immunological effect of PM2.5 on cytokine production in female Wistar rats. Methods Female Wistar rats were given 0.3 mg, 0.75 mg, 2 mg, 5 mg of PM2.5 per 0.5 mL saline, respectively. Saline was used as the negative control. TNF-α and IL-6 levels in the branchoalveolar lavage were measured by ELISA, and mRNA expression levels in lung tissue were detected by RT-PCR. Alveolar macrophages were collected for testing phogacytic function. Results Exposure to PM2.5 stimulated TNF-α production in a dose-dependent manner （P〈0.05）, However, no statistically significant difference was found. No time-dependent change in TNF-α and IL-6 production was found. TNF-α and IL-6 mRNA expressions were induced by PM2.5-exposure. The phagocytic rate （PR） was significantly decreased by PM2.5 treatment. Conclusion PM2.5 exposure increases inflammation response of the lung in a dose-dependent manner. Moreover, tissue injury induced by PM2.5 may be related to altered production of cytokines. PM2.5 may impair the phagocytic activity of alveolar macrophages.     

Antithrombin-III without concomitant heparin improves endotoxin-induced acute lung injury rats by inhibiting the activation of mitogen-activated protein kinase

Background Antithrombin-Ⅲ （AT-Ⅲ）, the major inhibitor of thrombin in plasma, also has anti-inflammation property and might have positive effect on sepsis. The present study aimed to investigate the effects of AT-Ⅲ on inflammatory reaction and pulmonary protection in endotoxin-induced acute lung injury （ALI） rat. Methods Sixty male Sprague-Dawley rats were randomly assigned equally to normal control group, ALl group, AT-Ⅲ treatment group, AT-Ⅲ＋heparin treatment group, and heparin treatment group. The pulmonary vascular permeability index （PVPI） was measured by single nuclide tracer technique. The activity of AT-Ⅲ in plasma was determined by the method of synthetic chromogenic substrata. Tumor necrosis factor-a （TNF-a） and interleukin-6 （IL-6） levels in serum were determined by enzyme-linked immunosorbent assay. The expressions of lung tissue mitogen-activated protein kinases （ERK1/2, P38 and JNK MAPK） were determined by Western blotting. Results Rats had significantly improved lung histopathology in the AT-Ⅲ treatment group and heparin treatment group compared with the ALl group, The PVPI of the ALl group was 0.38±0.04, significantly higher than that of the normal control group （0.20±0.02, P 〈0.01）, AT-Ⅲ treatment group （0.30±0.04, P 〈0.01） and heparin treatment group （0.28±0.04, P 〈0.01） respectively. There were no significant differences of PVPI in the ALl group and AT-Ⅲ＋heparin treatment group. The activity of AT-Ⅲ in plasma in the ALl group was （76±8）%, significantly lower than that of the normal control group （（96±11）%, P 〈0.05） and AT-Ⅲ treatment group （（105±17）%, P 〈0.05） respectively. The serum levels of TNF-α and I L-6 of the ALl group were （2.770±0.373） μg/L and （1.615±0.128） ng/ml respectively, significantly higher than those of the normal control group （0.506±0.093） μg/L and （0.233±0.047） ng/ml respectively, all P 〈0.01）, AT-Ⅲ treatment group （（1.774±0.218） pg/L and （1.140±0145） ng/ml respectively, all P 〈0.01） and heparin treatment group （（1.924±0.349） μg/L and （1.223±0.127） ng/ml respectively, all P 〈0.01）. The lung tissue levels of phospho-ERK1/2 and phospho-P38 MAPK expressions were markedly higher in the ALl group than in the normal control group, AT-Ⅲ treatment group and heparin treatment group respectively. Conclusions AT-Ⅲ without concomitant heparin inhibited the activation of ERK1/2 and P38 MAPK, down-regulated the levels of downstream cytokines TNF-a and IL-6, relieved endothelial permeability, and improved the ALl in endotoxin-induced rats. It might be helpful to administrate AT-Ⅲ alone, not with concomitant heparin, to those patients with ALl and sepsis.     

Effect of ARB on Expression of CD68 and MCP-1 in Adipose Tissue of Rats on Long-term High Fat Diet

In adipose tissue of rats on long-term high fat diet, the inflammatory changes the roles of angiotensin receptor blocker （ARB） in pimelitis and insulin resistance （IR） were observed. LR rat model was established by feeding high calorie and high fat diet. The change in insulin sensitivity was detected by euglycemic-hyperinsulinemic clamp technique 8 weeks after intervention by valsartan. The expression levels of CD68 and MCP-1 mRNA and proteins in adipose tissue were examined by RT-PCR and immunohistochemistry respectively. The parameters of blood glucose, insulin and blood lipid were analyzed. The results showed that in high fat diet group intra-abdominal obesity developed the content of visceral fat and the number of inflammatory cells in local adipose tissue were significantly increased （P〈0.01）, the levels of serum triglyceride, free fatty acids and fasting serum insulin were markedly increased, the insulin sensitivity was significantly lowered （P〈0.01）, and the expression of CD68 and MCP-1 was significantly increased as compared with control group （P〈0.01）. In ARB interventional group, the content of visceral fat, the number of inflammatory cells and the ex- pression of CD68 and MCP-1 in local adipose tissue were significantly reduced （all P〈0.01）, but the insulin sensitivity was significantly enhanced （P〈0.01） as compared with high fat diet group. There were pimelitis and IR in rats with obesity induced by long-term high calorie and high fat diet. The ARB can significantly inhibit the infiltration of macrophages and the expression of MCP-1 in adipose tissue, thereby attenuating the inflammation and improving LR in rats.     

Effect of Dietary Oleic Acid and Palmitic Acid on Cerebellum Histological Changes and Sensorimotor Coordination in Rat

Lipids are important constituents of the neuronal membrane. Also, kinds of fatty acids were supplied in the diet can affect the brain growth and the onset of myelinogenesis. So, in this study, the effect of oleic acid and palmitic acid on sensorimotor coordination and cerebellum histological changes was investigated. In present study, 9 groups （10 rats in each group） of Wistar immature rats were used. One group kept as control with similar environmental conditions to other groups. Four groups of rats were fed with diet containing 10% palmitic acid for 1 to 4 weeks respectively. Four groups of rats were fed with diet containing 10% oleic acid for 1 to 4 weeks respectively. After maturation （age at 90 weeks）, the rats were tested for motor activity task by using Rota rod based on standard method and then the whole cerebellum were removed and samples were fixed in bouin for routine paraffin embedding method. The results showed that sensorimotor coordination was significantly （p 〈 0.01） increased in rats fed with oleic acid for 4 weeks. The sensorimotor coordination was significantly （p 〈 0.01, p 〈 0.5） increased in groups received palmitic acid for 1 and 2 weeks. Also, diameter of cerebellum was significantly （p 〈 0.001, p 〈 0.5） changed with oleic acid for 4 weeks and palmitic acid for 2 weeks. The diameter of molecular layer of cerebellum was significantly （p 〈 0.001, p 〈 0.5） changed with oleic acid for 2 and 4 weeks. Thus, palmitic and oleic acid can improve function of tissue of cerebellum in rat.     

Effects of valsartan on diabetic cardiomyopathy in rats with type 2 diabetes mellitus

Background The development of diabetic cardiomyopathy is multifactorial. Insulin resistance （IR） and excessive activity of the renin-angiotensin system are confirmed reasons for diabetic cardiomyopathy. Renin-angiotensin system （RAS） inhibitors can reduce tissue Ang Ⅱ levels, with beneficial effects on cardiovascular function. Therefore, in type-2diabetes mellitus （T2DM）, blockade of the RAS may have the function of protecting against diabetic cardiomyopathy through increasing insulin sensitivity and inhibiting excessive activity of RAS. However, this has not been confirmed.Methods The effect of valsartan, an angiotensin receptor blocker （ARB）, on diabetic cardiomyopathy in the presence of T2DM was studied. Wistar rats with T2DM and T2DM treated with valsartan were studied. Glucose infusion rates （GIR）,index of IR, heart weight, the heart weight-to-body weight ratio （HW/BW）, myocardial apoptotic index, cardiac hydroxyprolin content, and cardiac tissue collagen type Ⅰ and collagen type Ⅲ content were measured.Results GIR in T2DM rats and T2DM rats treated with valsartan decreased （P ＜0.01）. In T2DM rats treated with valsartan, heart weight, myocardial apoptotic index, cardiac hydroxyprolin content, and cardiac tissue collagen type Ⅰ and collagen type Ⅲ content were higher than in control rats, but lower than in T2DM rats. In rats with T2DM, GIR was negatively and significantly correlated with all the variables. However, in T2DM rats treated with valsartan or normal control rats, none of the correlations was significant.Conclusions In the presence of T2DM, diabetic cardiomyopathy is related with IR. Valsartan can not alleviate IR, but can protect against diabetic cardiomyopathy and remove the correlation between IR and diabetic cardiomyopathy.     

Protective Effect of Genistein on Lipopolysaccharide-induced Acute Lung Injury in Rats

Summary： To investigate the protective effect of genistein on endotoxin-induced acute lung injury in rats, and explore the underlying mechanisms, 32 male Sprague-Dawley rats were randomly divided into 4 experimental groups： saline control, genistein alone, lipopolysaccaride alone, and genistein pretreatment. Each treatment group consisted of eight animals. Animals were observed for 6 h after LPS challenge, and the wet/dry （W/D） weight ratio of the lung and bronchoalveolar lavage fluid （BALF） protein content were used as a measure of lung injury. Neutrophil recruitment and activation were evaluated by BALF cellularity and myeloperoxidase （MP（）） activity. RT-PCR analysis was performed in lung tissue to assess gene expression of ICAM-1. The histopathological changes were also observed using the HE staining of lung tissue. Our results showed that lung injury parameters, including the wet/dry weight ratio and protein content in BALF, were significantly higher in the LPS alone group than in the saline control group （P〈0.01）. In the LPS alone group, a larger number of neutrophils and greater MPO activity in cell-free BAL and lung homogenates were observed when compared with the saline control group （P〈0.01）. There was a significant increase in lung ICAM-1 mRNA in response to LPS challenge （P〈0. 01, group L versus group S）. Genistein pretreatment significantly attenuated LPS-induced changes in these indices. LPS caused extensive lung damage, which was also lessened after genistein pretreatment. All above-mentioned parameters in the genistein alone group were not significantly different from those of the saline control group. It is concluded that genistein pretreatment attenuated LPS-induced lung injury in rats. This beneficial effect of genistein may involves, in part, an inhibition of neutrophilic recruitment and activity, possibly through an inhibition of lung ICAM-1 expression.     

Murine calcium-activated chloride channel family member 3 induces asthmatic airway inflammation independently of allergen exposure

Background Expression of murine calcium-activated chloride channel family member 3 （mCLCA3） has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin （OVA）. However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified. Methods mCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid （BALF） were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC （MUC5AC） were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 （IL-13） were determined quantitatively. Results mCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-y, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group （P 〈0.05）. The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALE The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue. Conclusion These findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.     

Yohimbine protects against endotoxin-induced acute lung injury by blockade of alpha 2A adrenergic receptor in rats

Background  Alpha 2A adrenergic receptor (AR) is a subtype of α2 AR belonging to G protein-coupled receptors, and exerts a variety of biological effects. Recent studies have demonstrated that the α2A AR activation was closely related with inflammatory reaction. The present study aimed to investigate the influence of α2A AR antagonist, yohimbine, on the severity of endotoxin-induced acute lung injury in rats.

Methods  A total of 72 male Sprague-Dawley rats were randomly divided into three groups: control group, lipopolysaccharide (LPS) group and LPS + yohimbine group. Rats were intratracheally administrated with normal saline or LPS (300 μg), and the rats in the LPS + yohimbine group were treated with additional yohimbine (2 mg/kg, i.p) soon after LPS administration. Six, 24 and 48 hours after treatment, arterial blood gas analysis was carried out, and optical microscopy was performed to evaluate pathological changes in the lung, and lung injury score was assessed. The count of white blood cells in bronchoalveolar lavage fluid (BALF) was determined. The levels of norepinephrine, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in BALF were measured with enzyme-linked immunosorbent assay. Immunocytochemistry was performed for the detection of α2A AR on inflammatory cells in BALF.

Results  When compared with the control group, the oxygenation index in the LPS group was significantly decreased, and white blood cell count, the lung histopathological scores, levels of norepinephrine and IL-6 as well as α2A AR expression on inflammatory cells in the BALF were dramatically increased at different time points, and the concentrations of TNF-α and IL-1β were also increased except at 48 hours after LPS administration. The oxygenation index decreased while white blood cell count in BALF and the lung histopathological scores were obviously increased in the LPS + yohimbine group. The level of norepinephrine in BALF was increased at each time interval in the LPS + yohimbine group, and so did the levels of TNF-α, IL-1β and IL-6 at 6 and 48 hours after LPS administration respectively. When compared with the LPS group, the oxygenation index, white blood cell count, the lung histopathological scores and the level of IL-6 in the LPS + yohimbine group were significantly improved at each time interval, and the concentrations of TNF-α and IL-1β were also lower at 24 hours of LPS administration (all P <0.05). Correlation analysis indicated the level of norepinephrine was related to the levels of TNF-α, IL-1β and IL-6 in the BALF and the lung histopathological scores (r=0.703, r=0.595, r=0.487 and r=0.688, respectively, P <0.001) and the intensity scores of immunoreactivity to α2A AR on inflammatory cells were also associated with the levels of TNF-α, IL-1β and IL-6 as well as the lung histopathologial scores (r=0.803, r=0.978, r=0.716 and r=0.808, respectively, P <0.001).

Conclusions  Yohimbine can inhibit TNF-α, IL-1β and IL-6 overproduction and relieve the severity of pulmonary inflammation induced by endotoxin, which is maybe mediated by blockade of α2A AR on inflammatory cells.

     

Matrix metalloproteinase-8 inhibitors mitigate sepsis-induced myocardial injury in rats

Background Sepsis-induced myocardial injury （SIMI） is caused by a variety of mechanisms. The aim of the study is to investigate the effects of metalloproteinase-8 （MMP-8） on SIMI and its mechanisms in rats. Methods Forty male Sprague Dawley rats were randomly divided into four groups： MMP-8 inhibitor （M81）, dexamethasone （DEX）, sepsis, and sham groups. The sepsis model was established by cecal ligation and puncture （CLP）. Rats in the M81 group immediately received an intraperitoneal injection of M81 （0.1 mg/kg） after CLP. Rats in the DEX group immediately received an intraperitoneal （IP） injection of DEX （2 mg/kg）. Rats in the sepsis and sham groups received intraperitoneal injections of normal saline. Rats were sacrificed 12 hours after CLP. Paraffin sections were stained with hematoxylin and eosin to observe the myocardium. The myocardial ultrastructure was observed with transmission electron microscopy. MMP-8, tumor necrosis factor-Q （TNF-a）, and interleukin-113 （IL-113） were detected by immunohistochemistry. The expression of MMP-8 was measured by Western blotting. TNF-a and IL-113 levels in serum and myocardial tissue were determined by enzyme-linked immunosorbent assay. Results Compared with the sham group, the myocardium in the sepsis group was seriously injured. MMP-8, TNF-α and IL-1β expression was higher in the sepsis group than in the sham group, Treatment with M81 or DEX, however, attenuated sepsis induced histopathological changes in the heart, and was associated with significant reductions in serum and myocardial levels of TNF-a and IL-113 （P 〈0.05）. M81 significantly inhibited MMP-8 expression in myocardial tissue （P 〈0.05）. In addition, treatment with DEX was not associated with a change in myocardial levels of MMP-8 （P 〉0.05）. Conclusion MMP-8 inhibitor attenuated myocardial injury in septic rats, which might be related to reduced expression of TNF-α and IL-1β.