| Background and ObjectiveChronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation, which is usually progressive.The disease is associated with an inhanced chronic inflammatory response in th airways and the lung to noxious and particles and gases. essentially, COPD is an airway inflammatory diseases, and the anti-inflammatory treatment is an important part of the treatment of COPD. Glucocorticoid (GC) is the most important and the strongest effective anti-inflammatory agent. In recent years, Although the application of GC in patients with COPD is increasingly widespread, the effect is not ideal, so there may be some mechanism in patients with COPD, which is resulted to reduce the anti-inflammatory effect of glucocorticoid, and that is glucocorticoid resistance. The Glucocorticoid resistance mechanisms is included the decrease of histone deacetylase2(HDAC2) activity, abnormal of glucocorticoid receptor, and the lack of NF-kappa B activation pathway. Among of them, the decrease of HDAC2the main reason of glucocorticoid resistance.The homeostasis of histone acetylase with HDAC regulates the activation and transcription of inflammatory genes, histone acetylase catalyzed the fifth Lysine residues (Lys) at the end of the core histone molecules acetylated histone, and make the histone with a negative charge, loosely binding with the negative charged chromatin, and the DNA appears depolymerization effects, transcription factors are recruited to promote the transcription of the inflammatory gene. HDAC can make the histone deacetylate, and the binding of Molecular with histone becomes closer, which blocks the gene transcription. HDAC2is a type of HDAC I, which exists only in the nucleus.It is the main subtypes involved in the pathogenesis of COPD, disincents the transcription and activationof gene. The study confirmed that the HDAC2expression and activity is decreased in the nucleus of the lung epithelial cells and inflammatory cells of COPD rats, which mainly due to the oxidative stress. Superoxide anion generated in the oxidative stress is combined with the nitric oxide generated in the nitrification stress which produces the peroxynitrite.It can nitrate the tyrosine residues of HDAC2, or activate the PI3K/AKT pathway, phosphorylating the serine residue of HDAC2, resulting the proteasome of HDAC2inactivate, and then decreased the activity of HDAC2. HDAC2is the intermediaries of anti-inflammatory effect of GC.With the activity of HDAC2decreased, the number of which can be recruited to the sites of gene expression is reduced, and then the degree of histone acetylation is enhanced, which increasing the expression of inflammatory gene, and the release of IL-8, TNF-α and other inflammatory factors is increased. The anti-inflammatory effect of GC’is weakened. So enhancing the activity of HDAC2is the important target for COPD glucocorticoid resistance.8-iso-prostaglandin F2a (8-iso-PGF2a) is the production of lipid peroxidation of arachidonic acid in the membrane by the oxygen free radicals. Its production is not dependented by enzymatic pathways and the chemical property is stable, therefore it is used as a ideal indicator of the evaluation of oxidative stress levels. At home and abroad,the8-iso-PGF2a is only limited to the correlation with the oxidative stress and inflammation level, the reports of the correlation with HDAC2are fresh.Erythromycin is a representative drug of macrolide antibiotics. In addition to having antimicrobial activity, it also has significant anti-inflammatory activity, antioxidant activity. It can inhibit the release of IL-8and the activation of neutrophil by inhibiting NF-κB, AP-1activity, and it can also reduce the reactive oxygen species,which is induced by macrophages in the role of cigarette smoke extract (CSE) in vitro. In addition, roxithromycin and azithromycin, which belong to the macrobiotic antibiotic like erythromycin can inhibit the activation of the PI3K/Akt signaling pathway. Furthermore, PI3K/Akt signaling pathway is the important pathway of oxidative stress to reduce HDAC2activity. Therefore, whether erythromycin is able to inhibit oxidative stress by inhibiting the activation of the PI3K/Akt signaling pathway,and enhance HDAC2activity, reversal of glucocorticoid resistance, which has been reported rarely at home and abroad.Budesonide is the second generation of adrenocorticotropic hormone,which can act directly through inhalation pathway and reduce airway mucosal inflammation.Whether erythromycin can improve the sensitivity of budesonide locally or systemic is not reported.In this study, COPD rat models were established by the way of twice airway instillation of lipopolysaccharide with passive smoking,Given erythromycin and budesonide based on the establish of COPD rats models,and the observate the effect of erythromycin on HDAC2activity and glucocorticoid resistance in COPD rats models, and to explore the role of the PI3K/Akt signaling pathway.Materials and Methods50male rats with sprague dawley, weighing200±20g. Randomly divided into two groups:the healthy control group (n=10), COPD group (n=40). COPD group:1st,14th days, given airway instillation of lipopolysaccharide.2-13days,15-30days, given passive smoking in the fumigation chamber. C group:1st,14th days, given airway instillation of saline,2-13days,15-30days given air in the fumigation chamber breathing.With the Model established, the COPD group was randomly divided into COPD model group, erythromycin group, budesonide group, erythromycin+budesonide group, n=10. COPD model group:From the31days, given a gavage of normal saline,1/2ml/only,2weeks. Erythromycin group:From the31days, given a gavage of erythromycin,1time/day,100mg/kg,2weeks, budesonide group:From the31days, given aerosol inhalation of budesonide,1/2ml/only for2weeks, erythromycin+ budesonide group:From the31days, given a gavage of erythromycin, which dose and time like the above, given aerosol inhalation of budesonide, the dose and time like above. The45th day, test the pulmonary function, and speciment blood and bronchoalveolar lavage fluid samples. The level of IL-8, TNF-α,8-iso-PGF2a in the serum and bronchoalveolar lavage fluid supernatant were test by ELISA; lung tissue was by HE staining to observe lung tissue structural changes; The detection of the expression of HDAC2, PI3K, p-AKT were by Western-blot and immunohistochemical.1light microscopy of lung tissue morphologyCompared with the healthy control group, tracheal and bronchial epithelial cells fall off of the rats of the COPD model group, with the bronchial smooth muscle thickening, inflammatory cell infiltration, collagen deposition, alveolar wall thinning, or even rupture fused into the bulla. The changes were in line with the trachea and lung histopathology of the human with COPD. The damage of lung tissue with rats of drug intervention groups is lighter than the M group. Among them, the damage of rats of erythromycin+budesonide group is the lightest.2the lung function changes of ratsRats of COPD model group, tidal volume (TV), peak expiratory flow (PEF),50%of tidal volume and peak expiratory flow rate (EF50) were1.25±0.73ml,18.49±3.24ml/s,1.38±0.21ml/s, which was significantly lower than in healthy control group[1.87±0.76ml,24.96±11.38ml/s,1.73±1.02ml/s], and the difference was statistically significant (P<0.001). The value of erythromycin group [1.68±0.54ml,23.30±10.34ml/s1.68±0.54ml/s], the value of the budesonide group [1.52±0.88ml,19.62±10.52ml/s,1.47±0.97ml/s], erythromycin+budesonide group [1.72±0.97ml,24.87±10.58ml/s,1.68±0.40ml/s] are higher than the COPD model group, and the difference was statistically significant (P<0.001).3the Level of8-iso-PGF2a in lavage fluid and serum8-iso-PGF2a of the lavage fluid and the serum of control healthy group has a value of19.10±0.91ng/ml, COPD model group [37.14±0.21ng/ml,40.74±6.86ng/ml], was higher than healthy control group, the difference was statistically significant (P<0.001); erythromycin group [19.62±0.76ng/ml,28.96±0.55ng/ml], the value in lavage of budesonide group [23.50±1.01ng/ml], erythromycin+budesonide group [19.38±0.74ng/ml,29.73±0.69ng/ml], were lower than COPD model group, and the difference was statistically significant (P<0.001); Although the value in the serum[38.14±3.37ng/ml]is lower than the control healthy group, the difference was not statistically significant (P=0.435);The budesonide group was significantly higher than the erythromycin group, and the difference was statistically significant (P<0.001); The expression with erythromycin group is lower than erythromycin+budesonide group, but the difference was not statistically significant (P=0.621,0.797); compared with budesonide group, expression levels were significantly lower, the difference was statistically significant (P<0.001).4the level of IL-8and TNF-a in serum and lavage fluidIn the lavage flude,IL-8, TNF-α of healthy control group has a value of63.79±3.42ng/L,110.39±9.38ng/L, COPD model group [127.70±9.32ng/L,280.81±4.48ng/L], was higher than the healthy control group, the difference was statistically significant (P<0.001).erythromycin group [64.97±3.05ng/L,117.59±10.71ng/L],budesonide group [80.31±3.71ng/L,170.78±12.51ng/L], erythromycin+budesonide group [65.47±2.29ng/L,116.86±6.64ng/L]were lower than the COPD model group, the difference was statistically significant (P<0.001); budesonide group was significantly lower than in the erythromycin group, the difference was statistically significant (P<0.001); erythromycin+budesonide group was significantly lower than in erythromycin group, but was no significant difference(P=0.787、0.902),It is lower than the budesonide group, the difference was statistically significant (P<0.001).In the serum,IL-8, TNF-α of healthy control group has a value of99.16±2.23ng/L,240.70±6.68ng/L, COPD model group [153.82±2.21ng/L,436.76±3.33ng/L], was higher than the healthy control group, the difference was statistically significant (P<0.001). erythromycin group [110.60±4.33ng/L,275.00±8.28ng/L], erythromycin+budesonide group [102.11±2.35ng/L,252.15±7.95ng/L]were lower than the COPD model group, the difference was statistically significant (P <0.001); budesonide group [134.97±2.67ng/L,398.68±7.71ng/L], was lower than the COPD model group, but was no significant difference (P=0.693、0.505); budesonide group was significantly lower than in the erythromycin group, the difference was statistically significant (P<0.001); erythromycin+budesonide group was significantly lower than in erythromycin group、budesonide group, and the difference was statistically significant (P<0.001).5lung tissue HDAC2, PI3K,p-AKT immune group:HDAC2is multi expressed in bronchial mucosal epithelial cells, vascular endothelial cells, alveolar epithelial cells and alveolar macrophages nucleus. PI3K, P-AKT expressed in bronchial epithelial cells and airway smooth muscle cells, and inflammatory cells. Combined with healthy control group, The HDAC2expression of COPD model group reduced, but PI3K%p-AKT expression enhanced, and the difference was statistically significant (P<0.001). compared with COPD model group, HDAC2expression intensity, PI3K、p-Akt expression is reduced of erythromycin group and erythromycin+budesonide group, the differences were statistically significant (P<0.001). Compared COPD model group group, HDAC2expression is enhanced, PI3K、p-Akt expression of budesonide group is reduced, but the difference was not statistically significant (P=0.145、0.203、0.178); Compared with erythromycin group, HDAC2expression intensity decreased, PI3K, p-AKT expression of budesonide group is significantly increased, the difference was statistically significant (P<0.001).Compared with erythromycin group, HDAC2enhanced, but expression of PI3K, p-AKT expression of erythromycin+budesonide group is weakened, the difference was not statistically significant (P=0.524、0.252); PI3K, p-AKT expression significantly decreased compared with the budesonide group, the difference statistically significant (P<0.001).6HDAC2, PI3K, p-AKT protein levels in lung tissue Compared with the healthy control group, HDAC2protein expression decreased, PI3K, p-AKT expression increased of COPD model group, the difference was significant (P<0.001). Compaired with COPD model group, HDAC2protein expression intensity, PI3K, p-Akt expression was significantly reduced of erythromycin group, erythromycin+budesonide group, and the difference was statistically significant (P<0.001). Compired with healthy control group, HDAC2expression enhanced, PI3K,p-Akt expression is reduced of budesonide group, but the difference was not statistically significant (P=0.423、0.706、0.694); Compired with erythromycin group, HDAC2expression strength is reduced, PI3K, p-AKT expression was significantly increased of budesonide group, and the difference was statistically significant (P<0.001). Compired with erythromycin group, HDAC2expression enhanced, PI3K, p-AKT expression was significantly weakened of erythromycin+budesonide group, but the difference was not statistically significant (P=0.525、0.533、0.464); compared with budesonide group, HDAC2expression enhanced, PI3K, p-AKT expression significantly decreased, The difference was statistically significant (P<0.001).7Correlation analysis between HDAC2activity in the lung tissue and8-iso-PGF2alpha, IL-8, TNF-a in the serum (B) and bronchoalveolar lavage fluid (F), PI3K, P-AKT activity in the lung tissueHDAC2protein levels in lung tissue and8-iso-PGF2a levels in serum and bronchoalveolar lavage fluid was negatively related, the Spearman correlation coefficient rB=-0.985, rF=-0.834(P<0.001). HDAC2protein in lung tissue and IL-8levels in the serum and bronchoalveolar lavage fluid was negative correlation rB=-0.974, rF=-0.886(P<0.001); HDAC2was negatively correlated with TNF-a level, rB=-0.856, rF=-0.841(P<0.001).HDAC2protein and PI3K protein expression in lung tissue was negatively correlated, rs=-0.98(P<0.001) and p-AKT protein was a negative correlation, the correlation coefficient rs=-0.94(P<0.001). Conclusion1. Instillation of lipopolysaccharide in the airway2times (1,14days) linking passive smoking for28days could successfully replicate COPD rat model.2. Oxidative stress, inflammation injury and glucocorticoid resistance present in COPD rats.Erythromycin can inhibit oxidative stress, inflammatory cytokine production, which combinated with budesonide can significantly improve its anti-inflammatory effect.3. HDAC2in the COPD lung tissue is decreased. Oxidative stress and the PI3K/AKT pathway may was involved in the negative regulation of HDAC2activity4. Erythromycin may be enhanced the inhibiting of PI3K/AKT pathway in the lung tissue of COPD, which can enhance HDAC2activity and reverse the glucocorticoid resistance. |
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