The Effect Of Clarithromycin Reduced IL-32 On COPD Rat Glucocorticoid Resistance And Its Mechanism

Interleukin-32(IL-32) was first discovered in 1992,a cytokine secreted by NK cells and epithelium,but then it has not made the obvious progress in the study,It was not officially named IL-32 until 2005,Is one of the newest cytokines discovered.The present study shows that IL-32 has abnormal expression in immune deficiency, ulcerative colitis, Krohn and rheumatoid.In respiratory diseases,IL-32 was also expressed in pulmonary tuberculosis, lung cancer, asthma and chronic obstructive pulmonary disease.The study proves that there exists INF-γ)-- IL-32-- interleukin-1 beta(IL-1β)positive feedback pathway in human body.In Chronic Obstructive Pulmonary Disease(COPD) patients,The pathway is activated and the expression of IL-32 is significantly increased;Domestic and foreign studies show that,nifedipine, simvastatin, budesonide and other drugs can inhibit The expression of asthma in IL-32,Thus play an anti-inflammatory role, reduce the number and severity of acute asthma attacks,Foreign study also showed that budesonide could also inhibit expression of IL-32 in COPD.Domestic and foreign research that IL-32 mainly through the mitogen activated protein kinase phosphorylation pathway, nuclear factor-k B( NF-kb) pathway, caspase pathway activation of downstream signaling pathways.Upregulation of tumor necrosis factor alpha, IL-1β, IL-8, expression of inflammatory cytokines,Involved in the inflammatory response of COPD.,The research has confirmed that the expression of INF-γ--IL-32--IL-1β by regulating the expression of the expression of the growth factor- beta(TGF-β), vascular endothelialgrowth factor(VEGF), etc, is involved in the airway remodeling of COPD and the formation of emphysema.Glucocorticoids because of their widespread effective anti-inflammatory effect, are the most important drugs for the treatment of COPD,But its efficacy is far less asthma,The cause of this result is glucocorticoid resistance,Early studies considered that the main cause of glucocorticoid resistance was A lot of data indicated that glucocorticoid receptors were detected in glucocorticoid and animal models and decreased expression of glucocorticoid receptor in glucocorticoid receptor in patients and animal models.The latest research shows that the human GR exists alpha and beta two subtypes.,GR is a target receptor for glucocorticoid effect.,GR beta is an endogenous antagonist of glucocorticoid effect.,In the patients with steroid resistant asthma and blood disease, the expression of GR was down regulated and the expression of GR was up-regulated.,So GRα/GRβ imbalance is the important reason for glucocorticoid resistance.A few studies have shown that INF-γ-- IL-32-- IL-1β through NF-kb/AP-1 pathway is involved in the expression of GRα and GRβ,GRα expression was up-regulated and the expression of GR was down-regulated, and it was involved in glucocorticoid resistance.Clarithromycin is a macrolide antibiotic, has been widely used in the treatment of stable COPD,Research shows that clarithromycin can reduce the patients with stable TNF- COPD alpha, IL-8, IL-6 expression of inflammatory cytokines,Inhibit lung inflammation, improve exercise endurance, improve lung function.Clarithromycin in COPD anti-inflammatory effect has been recognized,But about clarithromycin treatment mechanism in COPD, although less research at home and abroad,Clarithromycin through INF-γ--IL-32--IL-1β pathway regulating downstream inflammatory factor expression, and thus play a therapeutic role in chronic obstructive pulmonary disease(COPD).Clarithromycin through INF-γ--IL-32--IL-1β regulating NF-KB / activator protein-1(AP-1), effect on GRα and GRβ expression and reverse glucocorticoid resistance,There is no similar study at home and abroad,This study is intended to explore the clarithromycin downregulation of IL-32 expression on effect of glucocorticoid resistance in COPD rats and its mechanism.Materials and methodsHealthy male SD rats 50, weight 180±20g.Randomly divided into: blank group(10 only), model group(40 only).Model group: 1, 31, 61 days after tracheal intubation, intratracheal drip into lipopolysaccharide, the 2-30, 32~60, 62~90 days in smokes of passive smoking in the smoke box.The healthy control group: 1, 31, 61 days after tracheal intubation, intratracheal instillation of saline, the 2-30, 32~60, 62~90 days in smoked cigarette box in the air to breathe.After the construction of the model, the model group was randomly divided into 4 groups,COPD group, clarithromycin, budesonide group, treatment group, 10 rats in each group.COPD group: ninety-first days beginning to give saline irrigation, 1time/day, 2ml/only, a total of 30 days.The ninety-first day: Clarithromycin group was given clarithromycin orally, 1 times/day, 50mg/kg, a total of 30 days.Budesonide group: on the ninety-first day of budesonide inhalation, 1 times / day, 1mg/, a total of 30 days.The combined intervention group: the 91 day began to clarithromycin was administered by gavage, dose and time ibid, atomization inhalation of budesonide dose and time ibid.121 st days to detect lung function, leaving blood and alveolar lavage fluid,Detection of serum and bronchoalveolar using ELISA perfusion level of INF-γ、IL-32、TNF-α、NF-k B;The pathological changes of lung tissue after HE staining were observed in some lung tissues.,The expression of GRα and GRβ was detected by immunohistochemistry, Blot Western and RT-PCR in other lung tissues.Statistical analysis: all data were analyzed by SPSS17.0 software.Describe the data with the average of the standard deviation,The comparison between the two groups was t test, and the single factor analysis of variance was used in the comparison among the groups.Correlation analysis using Pearson correlation. Significance test level a = 0.05.Result1 the pathological changes of lung tissues in ratsCompared with the control group, COPD group rat tracheal and bronchial epithelial cells shedding, inflammatory cell infiltration, thickening of bronchial smooth muscle, collagen deposition, alveolar wall thinning, or even rupture fused into bullae of lung. The pathological changes of trachea and lung in patients with COPD were consistent.The destruction of lung tissue in rats of each drug intervention group was lighter than that of COPD group, and the rats in the combined intervention group were the lightest and the lung tissue was the lightest..2 Changes of pulmonary function in each group ratsThe expiratory forced capacity volume 0.3/forced vital capacity(FEV0.3 /FVC) in the COPD groupwas significantly lower than that in the blank group,The difference was statistically significant(P<0.05);Resistance inspiratory(Ri), resistance Re(expiratory) in the COPD group were significantly higher than the blank group, and the difference was statistically significant(P<0.05).Clarithromycin group,budesonide group FEV0.3/FVC were higher than that of group COPD,And the difference was statistically significant(P<0.05);Clarithromycin group, budesonide group Ri and Re were significantly lower than COPD group,The difference was statistically significant(P<0.05).Joint intervention group FEV0.3/FVC higher than group COPD, clarithromycin group and budesonide group,The difference was statistically significant,Joint intervention group Ri, Re was lower than that of COPD group, clarithromycin group and budesonide group,The difference was statistically significant(P<0.05).3 INF-γ,IL-32, TNF-α,NF-k B level in serum And Alveolar lavage fluidIn COPD group,INF-γ,IL-32, TNF-α,NF-kB level in serum were significantly higher than those in the blank group, and the difference is statistically significant(P < 0.05);in COPD group, INF-γ,IL-32, TNF-α,NF-k B level in Alveolar lavage fluid was significantly higher than the control group,And the difference was statistically significant(P < 0.05).In Budesonide group and clarithromycin group,INF-γ,IL-32, TNF-α,NF-k B level in serum lower than that in group COPD,And the difference was statistically significant(P < 0.05);Budesonide group and clarithromycin group INF-γ,IL-32, TNF-α,NF-k B level in Alveolar lavage fluid was significantly lower than that of COPD,And the difference was statistically significant(P < 0.05).Joint intervention group, INF-γ,IL-32, TNF-α,NF-k B level in serum was significantly lower than that in the COPD group, budesonide group, clarithromycin group,And the difference is statistically significant(P < 0.05),the combined intervention group, INF-γ,IL-32, TNF-α,NF-k B level in Alveolar lavage fluid was significantly lower than that in the COPD group, the budesonide group, clarithromycin group,And the difference was statistically significant(P < 0.05).4 Immunohistochemistry of GRα and GRβ inlung tissuesThe expression of GRα in COPD group was significantly lower than that in the blank group,The difference was statistically significant(P < 0.05);GRβ expression was significantly higher than the blank group,The difference was statistically significant(P < 0.05).The expression of GRα in budesonide group, clarithromycin group were higher than that of group COPD,The difference was statistically significant(P < 0.05);The expression of GRβ was lower than that of COPD group,The difference was statistically significant(P < 0.05).Combined intervention group GRα expression is higher than that of COPD group, budesonide group, clarithromycin group,The difference was statistically significant(P < 0.05);The expression of GRβ was lower than that of COPD group, budesonide group, clarithromycin group, the differences were statistically significant(P < 0.05).5 GRα, GRβ protein expression level in the lung tissueThe expression of GRα protein in COPD group was significantly lower than that in the blank group,The difference was statistically significant(P < 0.05);The expression of GRβ protein was significantly higher than that of the blank group, and the difference was statistically significant(P < 0.05).The expression of GRα protein in budesonide group, clarithromycin group were higher than that of group COPD,The difference was statistically significant(P < 0.05);The expression of GRβ protein was lower than that of COPD group,And the difference was statistically significant(P < 0.05).Combined intervention group GRα protein expression levelex is higher than that of COPD group, budesonide group, clarithromycin group,The difference was statistically significant(P < 0.05);The expression of GRβ protein level was lower than that of COPD group, budesonide group, clarithromycin group,The difference was statistically significant(P < 0.05).6 mRNA levels of GRα and GRβ in lung tissuesThe levels of GRα m RNA in COPD group were significantly lower than that in the blank group,The difference was statistically significant(P < 0.05);GRβ m RNA levels were significantly higher than the blank group,The difference was statistically significant(P < 0.05).Budesonide group, clarithromycin group GRα m RNA levels were higher than that of group COPD,The difference was statistically significant(P < 0.05);The expression of GRβ m RNA was significantly lower than that of COPD group(P < 0.05).Joint intervention group GRα m RNA levels higher than the COPD group, budesonide group, clarithromycin group,The difference was statistically significant(P < 0.05);The expression of GRβ m RNA was lower than that of COPD group, budesonide group, clarithromycin group,The difference was statistically significant(P < 0.05).7 Correlation between INF-γ, IL-32,TNF-α, NF-kb in serum(B) and alveolar lavage fluid(F) and FEV0.3/FVC, GRα, GRβINF-γ were positively correlated with IL-32 in both serum and alveolar fluid of rats(P < 0.05); IL-32 were positively correlated with TNF-α,NF-kb in both serumand alveolar fluid of rats(P < 0.05);IL-32 in serum and alveolar lavage fluid were both negative correlatied with FEV0.3/FVC(P < 0.05).IL-32,TNF-α,NF-kb in serum and alveolar lavage fluid were both negative correlatied with immunohistochemistry, protein expression,m RNA expression of GRα(P < 0.05). IL-32,TNF-α,NF-kb in serum and alveolar lavage fluid were both positively correlatied with immunohistochemistry, protein expression,m RNA expression of GRβ(P < 0.05).Conclusion1. INF-γ-- IL-32-- IL-1β positive feedback loop is involved in the pathogenesis of COPD,Clarithromycin, budesonide alone or in combination both can inhibit the activation of the feedback loop, reduce the inflammatory reaction of the lungs and the whole body in COPD rats, and improve the pulmonary function of COPD rats.The expression of GRα in COPD rats decreased significantly,GRβ expression increased significantly,GRα/GRβ imbalance caused glucocorticoid resistance in COPD rats.3.clarithromycin,budesonide can reverse GRα /GRβ imbalance,reversal glucocorticoid resistance in COPD rats,The effect is more obvious when combined,This process is related to the INF-γ--IL-32--IL-1β positive feedback ring.