The Correlation Between The Expression Level Of Cofilin-1in PBMCs Of SRA And The Response To Glucocorticoid Therapy

Background:Bronchial asthma is a common chronic airway inflammatory and allergic disease. Glucocorticoids exert potent anti-inflammatory and immunosuppressive effects, which are highly recommended for manegement of persistent asthma as the preferred therapy for control of airway inflammation, exprecially after revoluting inhaled glucocorticoids. Inhaled GCs has been rapid development and most widely used in clinical because of their simple, qiuck in effect and less adverse effect. Due to systemic and serious side effects, oral or intravenous GCs are only used to alleviate acute onset of severe asthma.Different bronchial asthmatic patients respond differently to glucocorticoid therapy, and it has great variation between individuals. The most majority of patients are controlled symptoms and prevented exacerbations after oral or inhaled GCs. Howevers, a subset of asthmatic subjects do not respond to steroid therapy. The symptoms and lung function will not be significantly improved, even if they exposure to higher doses or long-term glucocorticoid treatment, and that can only lead to serious side effects. For research purposes, these subjects showing no response to GCs are defined as steroid-resistant asthma(SRA). SRA was first reported in1968by Schmart Z in treating six asthmatic patients with high-dose of GCs, but there was not apparent improvement of symptoms. From a clinical point of view, Stephen and colleagues thinked that SRA was defined as a failure of FEV1%to improve from a baseline value of≤75%predicted by≥15%after14days of treatment with40mg prednisolone orally, despite demonstrating>15%reversibility to an inhaled β2agonist. This diagnostic criteria is gradually being accepted internationally. SRA is belonging to refractory asthma and at a lower rate of prevalence, and individuals with SRA are likely to use more emergency medical care and require more hospital admission than patients well-controlled. The prevalence of SRA accouts for about0.01%-0.1%in foreign at first, and the rate is increasing to about10%now, but that is not clear in domestic. In order to reduce the cost, enhance the effect of healthcare resources on these special patients, and minimize the side effects of oral gulcocortioid, it is urgent to find some factors which can assess the response to glucocorticoid therapy.Cofilin-1was a actin-binding protein which ubiquitously expressed in eukary otic cells. There are three isoforms of protein, ADF, cofilin-1(non-muscle cofilin) and cofilin-2(muscle cofilin) in human, belonging to actin depolymerizing factor family. ADF/cofilin is involved in many different cellular processes that are essential for growth, differentiation, division, membrane organization and motility. The combination of cofilin and actin filaments changes the distortion of filaments, thereby promoting the separation and depolymerization of filaments. In addition, recent studies have shown that cofilin-1represented a novel factor that could cause glucocorticoid resistance by analysis of Hela cells. Clfilin-1could affect the distribution of glucocorticoid receptor (GR) on the subcellular, by increasing the nuclear fraction of GR to inhibit its activity. Overexpression of cofilin-1not only breakdown the cytokeletal, but also decreased the function of glucocorticoid by up-regulating of c-JUN. Nisha Vasavda and his colleagues have suggested that the expression of cofilin-1was significantly greater in severe glucocorticoid-insensitive asthmatic CD4+T cells than in those isolated from glucocorticoid-sensitive subjects, furthermore dexamethasone didn’t affect the expression of cofilin-1in cultived Jurkat T cells in vitro, and the overexpression of cofilin-1could reduce dexamethasone supression of inflamationary cytokine release. In addition, Cofilin-1can promote T lymphocyte chemotaxis. The progression of asthma is closely associated with the activation of TH2cells which can release IL-33. IL-33, binding to ST2L which is specific expressed on the surface of TH2cells, promotes the related inflammatory procession of TH2cells. Prefontaine, D has discovered that IL-33expression increased in bronchial biopsies from subjects with asthma compared with controls, as well as subjects with asthma severity and IL-33might be a novel inflammatory marker of severe and refractory asthma.However.they didn’t report the change of cofilin-1between before and after glucocorticoid treatment in bronchial asthmatic patients. According to their vitro experient, whether cofilin-1could cause GC resistance in vivo in asthmatic patients, or be a predictor to response to glucocorticoid therapy, all of these are worthy of further study. So we collect bronchial asthma from outpatient in Nanfang Hospital, all selected patients were treated according to new GINA. SRAs were selected according to the international diagnostic criteria. We compared the expression of cofilin-1in PBMCs of different asthmatic patients before and after treatment with40mg/d of oral prednisone to acknowledge whether cofilin-1could be a predictor for the response to glucocorticoid therapy. Part of glucocorticoid resistant mechanisms are also studied.Objectives:This study elevates the response to glucocorticoid therapy on asthmatic patients to understand the prevalence of SRA in guangzhou, and analyze the correlation between the expression level of cofilin-1in their peripheral blood and the response to glucocorticoid therapy. Part of glucocorticoid resistant mechanisms are also studied, such as the overexpression of GRβ and IL-33.Methods:Out-patients with asthma were collected in the Respiratory of Nanfang Hospital in Guangzhou from August2008to December2010. Subjects with asthma were given strict diagnoses according to the new criteria of the GINA.375cases were collected. We collected their personal history, occupational history and family history information. They compeleted the Asthma Control of Life Questionnaire (ACQ5) by introduction and were recorded the24h symptom scores of follow-up days. Only part of the patients were selected to participate in the experiment. All selected asthmatic patients were measured in pulmonary function, they have a characteristic of less than75%FEV1%and positivess of bronchodilation test. According to the response to glucocorticoids and the improvement of FEV1%after two weeks of oral glucocorticoids, patients were subdivided into two groups with Steroid-resistant asthma(SRA,8cases) and Steroid-sensitive asthma(SSA,23cases).All subjects consented this research. Before treatment,9ml peripheral venous blood were collected,2ml was used to test the percentage of eosinophils and neutrophils in peripheral blood, the next5ml was used to isolate PBMCs to detect the expression of cofilin-1and GR(3, another2ml was used to test the level of plasma IL-33. Their induced sputum were selected to make a count of different cells. After treatment,5ml peripheral venous blood were also collected, being used to isolate PBMCs to detect the expression of cofilin-1and GRβ.The statistical data was analyzed by SPSS13.0. Datas were reported as means±SEM and Medians. We compared the characteristics of subjects using the Student’s t test and Pearson’s chi-square test. Because the data of the expression of cofilin-1,GRβ, IL-33do not meet the normal distribution, thus we used the non-parametric test (Mann-Whitney U Test and Wilcoxon test). Compare the sample rate and population rate by Binomial Test. Analyze the correlation between the indicators of lung function(FEV1%and PEF%) and the expression level of cofilin-1, the different percentage of cells count and the expression level of cofilin-1by rank correlation analysis (Spearman). The significance level setted at P<0.05.Results:1. We collected375asthmas and we have selected23cases of SSA and8cases of SRA. The prevalence of SRA was about2.1%(8/375), lower than10%of overseas(Binomial Test, P=0.000).2. There were no significant difference between the age of the two groups (t=-0.055, P=0.957), the gender of the two groups(P=0.698), the course of disease (t=-0.297, P=0.769), the ACQ5scores, the scores of daytime-symptomand the scores of night-symptomof the two groups(t=-1.038,P=0.308; t=0.293,P=0.772; t=-0.635, P=0.531)3. There were no significant difference between the two groups about the FEV1%levels before treatment (62.7(40-70.3)vs44.8(41-51.2),Z=-1.851,P=0.064). SSAs had a significant improvement of FEV1%after two weeks of oral prednisone (62.7(40-70.3)vs78.4(67.4-84.3),Z=-4.198,P=0.000), but the levels of FEV1%didn’t significantly change in SRAs (44.8(41-51.2)vs41.8(31.2-52.3),Z=-0.210, P=0.833)4. There were no significant difference between the two groups in the count of blood eosinophils(Z=-1.521, P=0.128). the percentage of blood eosinophils(Z=-1.791, P=0.073) and neutrophils(Z=-0.711, P=0.477), the percentage of sputum eosinophils(Z=-0.906, P=0.365). But the percentage of sputum neutrophils was higher in SRAs than that of SSAs(Z=-3.216, P=0.001).5. Cofilin-1protein expression was greater in PBMCs of SRA than that of SSA before oral prednisone treatment (Cofilin-1/β-actin ratio1.82(1.29-2.24)vs1.21(1.00-1.29),Z=-2.884,P=0.004). The expressional level decreased significantly in SSA patients(1.21(1.00-1.29)vs0.67(0.46-0.97), Z=-4.045,P=0.000), but there was no obvious decrease in SRAs (1.82(1.29-2.24)vsl.51(1.24-1.96), Z=-1.680,P=0.093)6. The expression level of cofilin-1before treatment was negatively correlated with the improvement of FEV1%(r=-0.397, P=0.027). There was no significant correlation between the expression level of cofilin-1and the the percentage of blood eosinophils(r=0.251, P=0.182), neutrophils(r=0.074, P=0.697) and the percentage of sputum eosinophils(r=-0.217, P=0.276) separately, but there was a positive correlation between the baseline expression level of cofilin-1and the percentage of sputum neutrophils(r=0.439, P=0.022). The change of cofilin-1expression level was positively correlated with the increased level of PEF%(r=0.37, P=0.044) before and after treatment, but the degree of correlation was not significant.7. Most PBMCs from SSA subjects expressed GRP negatively with our detection methods before and after treatment, the expression of GRP in PBMCs of SRAs was higher than that of SSAs, and its expression was significantly ((GRβ:β-actin ratio)12.83*10-3vs216.4*10-3, Z=-2.521, P=0.012) increasing after oral prednisone.8. IL-33(pg/ml) expression was slightly higher in plasma of SRAs than that of SSAs before oral prednisone treatment(87.00(71.57-110.54)vs72.76(61.23-82.76),Z=-2.032, P=0.042). Conclusions;1. Compared with overseas, the prevalence of steroid resistance is lower in asthmatic population in guangzhou.2. The technology of induced sputum plays an important role in the diagnosis of airway inflammation, and the inflammatory phenotype of SRA may be neutrophilic one.3. The expression level of cofilin-1in patients with asthma was associated with the response to glucocorticoid therapy, and cofilin-1could be a predictor for the response to glucocorticoid therapy.4. GRβ and IL-33may be involved in the procession of gucocorticoid insensitivity, and they could provide new therapeutic targents for SRA.5. The mechanism of the resistant to steroid may be that cofilin-1can promotes GR transfering from cytoplasm to nuclear, affecting gene transcrption lead overexpression of GR(3. Cofilin-1plays an important role in the chemotaxis of neutrophils. The chemotaxis of neutrophils degranulate and produe inflammation factors. Also cofilin-1regulates immune function by affecting T-cells chemotaxis. The situation of enhanced activation of Th2cells, sustained releasing associated factors such as IL-33. All of these could result in low response to steroid.