| Objectives:T helper lymphocytes differentiate into two subsets , Th1 and Th2, each with distinct functions and cytokine profiles. IFN-γis the signature cytokine of Th1 cell. IL-4 is the corresponding signature cytokine of Th2 cell, which also secrete IL-5, IL-6, IL-9 and IL-13, but doesn't secrete IFN-γ. These two subgroups of helper T cells arise in response to different immunogenic stimuli and cytokines, and they constitute an immunoregulatory loop, cytokines from Th1 cells inhibit Th2 cells, and vice versa. An imbalance in this reciprocal arrangement may be the key to asthma: there is credible evidence that, a hypothesis of the imbalance between Th1 and Th2, higher levels of Th2 cytokine IL-4 , IL-5 and IL-13 and lower levels of Th1 cytokine IFN-γ, is prominent in the pathogenesis of asthma. More recent studies have found T-bet and GATA-3 may be "master regulators" of Th lineage determination when IL-12 and IL-4 can strongly drive differentiation of Th1 and Th2 cells, respectively. CpG ODN containing unmethylated cytosine guanine dinucleotides CG (CpG)motifs could induce Th1-like immune reaction and suppress Th2-like immune response. It is well known that airway remodeling is one of important patho-character in asthma, there is acidophilic cell and other inflammatory cell infiltration .Acidophilic cell can induce epithelium damage and abscission , post-injury epithelium can release fibroblast activation cytokine ,then precipate sub-epithelium fibroblast accumulation and activation. The maily function of post-injury epithelium is that it can induce the sub-epithelium fibroblast swithing to myofibroblast, myofibroblast can expressα-SMA(α-smooth muscle actin),it is one kind of sub-phenotype in myofibroblast,it have contract function and strongly collagen synthesis ability. Myofibroblast is the critical cell to form the collagen in lung, the accumulation of myofibroblast promote the extracellular matrix excessively deposition .especially type I collagen; The high expression ofα-SMA advance the brochi contraction ,at the same time , type I collagenlet the bronchi stiffness and thicking,both of them are the key point in airway remodeling of asthma.Therefore,the expression of type I collagen andα-SMA can evaluate the degree of airway remodeling. Credible evidence has confirmed that the low level T-bet mice and the high level GATA3 mice display the similar allergy and the airway remodeling .Therefore, T-bet/GATA-3 ratio imblanced theory is regard as one of the major causes about sub-epithelium fibrosis and collagen deposition in the airway remodeling.Our study aimes to investigate the relationship between T-bet/ GATA-3 ratio in PBMCs and the imbalance of Th1/ Th2 cells from patients with asthma and to observe effect of CpG ODN on the ratio of T-bet/ GATA-3 and the imbalance of Th1/ Th2 cells, we examine the role of T-bet/GATA -3 ratio mRNA and protein in asthma model ,then focus on theα-smooth muscle actin (α-SMA)of pulmonary branchi and type I collagen in the lung fibroblast(LF) which are the mainly signature index of airway remodeling .Therefore,we find the balance point of T-bet/GATA-3 ratio to reverse the imbalance of Th1/ Th2 and interrupt the advancement of airway remodeling in earlier, eventually provide more effective methods for the treatment of asthma's patients.Methods:Peripheral venous blood 6ml were obtained from 30 cases with exacerbating asthma (asthma group) and 20 cases with chronic obstructive pulmonary disease (COPD group) and 20 normal cases (control group) ,each to add EDTA 0.8ml for anticoagulation. The plasma were isolated by centrifugation and stored in -80℃. PBMCs were isolated by density gradient centrifugation. The expression of T-bet mRNA and GATA-3 mRNA in PBMCs were measured by reverse transcription-polymerase chain reaction (RT-PCR) and the plasma levels of IFN-γ, IL-4 , IL-5 and IL-13 were measured by Enzyme-linked immunoadsordent assay (ELISA) .Peripheral venous blood 10ml were obtained from 20 cases with exacerbating asthma, each to add EDTA 1.2ml for anticoagulation. PBMCs were isolated by density gradient centrifugation and incubated with phytohemagglutinin (PHA) in vitro for 48h in the absence (blank group) or presence (CpG group) of CpG ODN.Supernatant and sediment were collected, respectively. Expression of IFN-γ, IL-4 , IL-5 and IL-13 in supernatant were determined by ELISA. The expression of T-bet mRNA and GATA-3 mRNA in sediment were measured by RT-PCR.Pathogen-free 30 rats (wistar) (the Shandong University experiments animal center, the SPF level, 6-8 wk old)were set in the identical raising room, the free potable water and food. They were divided into 3 groups : blank group , saline group and OVA group and matched for age ,sex and weight for studies.The concentration of T-bet mRNA and the GATA-3 mRNA in the lung were measured by the Quantitative Real-time PCR , the T-bet and GATA-3 protein were measured by western blotting. Lung fibroblast of each group were isolated as described above,the expression of type I collagen in the LF were determined by ELISA. Theα-SMA expression level were measured by immunohistochemistry .Meanwhile, comparing the relationship among T-bet mRNA /theGATA-3 mRNA,α-SMA and type I collagen.Data were analysed using the Statistical Package of the SPSS 10.0. Discriptive data were given as means±SD. Continuous variables were tested by analysis of t-test and the relations between the variables were tested by linear correlation. The p-values are two-tailed and a p-value of less than 0.05 being considered to be significant. The p-values are two-tailed and a p-value of less than 0.01 being considered to be more significant. Results:1. The T-bet mRNA expression in PBMCs in asthma group was significantly attenuated than those in COPD group and control group (P <0.05), meanwhile the GATA-3 mRNA expression was significantly enhanced (P <0.05), respectively. The T-bet mRNA expression in PBMCs in COPD group was enhanced than that in control group (P >0.05). The GATA-3 mRNA expression was attenuated (P >0.05) , respectively.2. The plasma levels of IL-4, IL-5 and IL-13 in asthma group were significantly higher than those in COPD group and control group (P <0.05) , respectively. The plasma level of IFN-γin asthma group was significantly lower than that in COPD group (P <0.05) , and was lower than that in control group(P>0.05) .3. The plasma level of IFN-γin COPD group was significantly higher than those in asthma group and control group (P <0.05), meanwhile the plasma level of IL-13 was significantly lower (P <0.05), respectively. However, the plasma levels of IL-4 and IL-5 were lower than those in asthma group and control group (P>0.05) , respectively.4. The ratio of T-bet/GATA-3 in asthma group was significantly lower than those in the COPD group and control group (P <0.05) . The ratio of T-bet/ GATA-3 in COPD group was higher than that in control group (P >0.05) .5. The T-bet mRNA expression has positive correlation with the plasma level of IFN-γ(r=0.675) (P<0.05) , has negative correlations with the plasma levels of IL-4 (r=-0.780), IL-5 (r=-0.664), and IL-13 (r=-0.546) (P<0.05) ,respectively.6. The GATA-3 mRNA expression has negative correlation with the plasma level of IFN-γ(r=-0.446) (P <0.05 ) , has positive correlations with the plasma levels of IL-4 (r=0.518 ), IL-5(r=0.393), and IL-13 (r=0.350) (P<0.05),respectively.7. The ratio of T-bet/GATA-3 has positive correlation with the plasma level of IFN-γ(r=0.833 ) (P <0.05 ) , has negative correlations with the plasma levels of IL-4 (r=-0.779), IL-5 (r=-0.631), and IL-13 (r=-0.584) (P <0.05) , respectively.8. The supernatant levels of IL-4, IL-5 and IL-13 in CpG group were significantly lower than those in blank group (P <0.05) , respectively. The supernatant level of IFN-γin CpG group was significantly higher than that in blank group (P<0.05) .9. The T-bet mRNA expression in sediment in CpG group was significantly enhanced than that in blank group (P <0.05) ,and the GATA-3 mRNA expression was significantly attenuated (P <0.05), respectively. The ratio of T-bet/GATA-3 in sediment in CpG group was significantly higher than that in blank group (P <0.05) .10. The T-bet mRNA expression in OVA group was significantly attenuated than those in blank group and saline group ( P=8.317×10-8 ,P=8.487×10-6) ;meanwhile the GATA-3 mRNA expression was significantly enhanced (P= 2.2×10-16,P=2.3×10-16)respectively;The ratio of T-bet/GATA-3mRNA in OVA group was significantly lower than those in blank group and saline group (P = 7.748×-07,9.645×-06 ) ;The saline group and blank group were no difference(P=0.1307,P=0.605,P= 0.056) .11. The T-bet protein expression in OVA group was significantly attenuated than those in blank group and saline group (P= 1.433×10-14 ,P= 4.926×10-7 ) ;meanwhile the GATA-3 protein expression was significantly enhanced (P=2.149×10-10,P=4.409×10-9) respectively;The ratio of T-bet/GATA-3 protein inOVA group was significantly lower than those in blank group and saline group (P = 2.287×-08,2.969×-06). The saline group and blank group were no difference(P=0.4987 ,P= 0.6077, P= 0.7859).12. Theα-SMA expression in OVA group was significantly enhanced than those in blank group and saline group (P= 8.447×10-3 ,P= 7.883×10-3 ).13. The expression of the type I collagen in LF was significantly enhanced than those in blank group and saline group (P= 1.603×10-6, P=1.458×10-6) .14. The ratio of T-bet/GATA-3 mRNA and protein have negative correlations withα-SMA (r=-0.8274,r=-0.9783) (p=3.742×10-6 ,p=6.905×10-6); The ratio of T-bet/GATA-3mRNA and protein have negative correlations with type I collagen (r=-0.779, r=-0.9449)) (P=3.811×10-8,P= 1.603×10-6) .Conclusions:1.The ratio of T-bet/ GATA-3 is the important evaluation index for the imbalance of Th1/Th2 cells.2.CpG ODN could influence the ratio of T-bet/GATA-3 by upregulate the T-bet mRNA expression and downregulate the GATA-3 mRNA expression , so that could inhibite the inflammatory response of asthma in upstream level.3.The ratio of T-bet/GATA-3 is the maily factor to induce the theα-SMA and type I collagen high expression which is the key point to airway remodeling of asthma.
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