Study The Effect Of The Proliferation, Apoptosis And The Synthesis And Secretion Were By Toll Like Receptor 4 In Asthmatic Rat Asmcs

Background Bronchial asthma (the asthma) is a airway disease of chronic inflammation that combination of cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components . Its important pathological feature of chronic airway inflammation and airway remodeling. Chronic inflammation is an immune response that is consist of multi-cellular and cellular components . Characteristic of Airway remodeling are airway smooth muscle cells hypertrophy, mucus gland hyperplasia, basement membrane thickening, mucosal metaplasia and hyperplasia of new blood vessels .However, the mechanism of airway inflammation and airway remodeling is not clear entirely today. In recent years, studies have shown that ASMCs not only involved in airway remodeling as target cells, but also involved in airway inflammation as the immune cells. So, Airway smooth muscle cells is an important role in the occurrence and development in asthma. Toll-like receptor (Toll like receptors, TLRs)was discovered in recent years which is a innate immune receptors of highly conserved and ancient family, which mediate innate immunity and acquired immunity, it can contribute to a variety of inflammatory cytokines and cytokine release, trigger, and increased inflammatory response. Overseas research shown Toll-like receptor 4 (TLR4) is expressed in airway smooth muscle cells, as the same time confirmed tant the ligand of Toll-like receptor 2 (TLR2) and TLR4 induced the release of cytokines, chemokines in airway smooth muscle cells, in the conditions of peripheral blood mononuclear cells and normal airway smooth muscle cells are cultured together.Howver. Domestic no reports ahout the relationship of TLRsand ASMCs. Overseas research confined the relationship between the TLRs of the normal airway smooth muscle cell-surface was activated and the secretory function , not addressed study on the role of TLRs in a state of asthmatic ASMCs.Sraat and others confirmed ActivatedTLR4 can promote the proliferation of vascular smooth muscle cells,Airway smooth muscle cells and vascular smooth muscle cells are belong to the smooth muscle cells , their morphology and functionare very similar .So we speculate a number of inflammatory mediators, cytokines in asthmatic airway can activate TLRs in the membrane of ASMCs ,TLRs may play an important role in the process of occurrence and development in asthma by combine different ligand and induce airway smooth muscle cells to produce inflammatory responses, the release of inflammatory mediators and promote cell proliferation, inhibit apoptosis.Summary, We will establish asthmatic rat model and take lung tissue and use image analysis software measurement of airway wall thickness and separation of airway smooth muscle cells and culture airway smooth muscle cells; Study on the impact of TLR4 in airway smooth muscle cells proliferation and apoptosis and synthesis and secretion in asthma by using of RNAi technology, RT-PCR, Western blot, TUNNEL, ELISA and other methods.Methods We used cell culture, immunohistochemical staining (IHC), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay (ELISA), MTT minim colorimetry and TUNNEL techniques, and adopted TNF-α, NF-ΚB inhibitor pyrrolidine dithiocarbamate(PDTC) as tool drugs, to study whether TLR4 regulates the cells proliferation and apoptosis and synthesis and secretion, the expression of TLR4 in ASMCs in vitro; We also establish asthmatic rat model ,delected the expression of TLR4 protein and mRNA by Western blot and RT-PCR, delected the expression of proliferative cell nuclear antigen (PCNA) and Bcl-2 protein by IHC and measured the airway wall thickness (WA / pi), bronchial smooth muscle thickness (smooth muscle area / pi), the number of bronchial smooth muscle nucleus (N / pi) by image analysis system to study the role of TLR4 in the asthmatic airway remodeling.1.Results(1)The proliferation of ASMCs in Asthma group was significantly higher than control group respectively(p<0.01); The expression of IL-5. IL-8 in supernatant in Asthma group was significantly higher than control group.(2) the best time of siRNA transfected asthmatic airway smooth muscle cells is 24h by the results of RT-PCR analysis.2.1 Results(1)The proliferation of ASMCs in siRNA-TLR4 transfction group was lower than control group(p<0.05); The proliferation of ASMCs in TNF-αgroup was significantly higher than control group and siRNA-TLR4 transfection group and TNF-α+ siRNA-TLR4 transfection group respectively(p<0.01). (2)The apoptosis rate of ASMCs in siRNA-TLR4 transfection group and TNF-α+ siRNA-TLR4 transfection group were significantly higher than control group(p<0.01). The apoptosis rate of ASMCs in TNF-αgroup was lower than control group and siRNA-TLR4 transfection group and TNF-α+ siRNA-TLR4 transfection group respectively(p<0.01).(3)The expression of IL-5. IL-8 in supernatant in control group and TNF-αgroup were significantly higher than TNF-α+ siRNA-TLR4 transfection group and siRNA-TLR4 transfection group(p<0.01); The expression of IL-5. IL-8 in TNF-α+ siRNA-TLR4 transfection group was significantly higher than siRNA-TLR4 transfection group (p<0.01); The expression of IL-5. IL-8 in TNF-αgroup was significantly higher than control group and TNF-α+ siRNA-TLR4 transfection group and siRNA-TLR4 transfection group(p< 0.01).(4)The mRNA and protein expression of TLR4 in control group and TNF-αgroup were significantly higher than siRNA-TLR4 transfection group and TNF-α+ siRNA-TLR4 transfection group(p<0.01); The mRNA and protein expression of TLR4 in TNF-αgroup was significantly higher than control group(p<0.01); There was no significant difference in the mRNA and protein expression of TLR4 between the siRNA-TLR4 transfection group and the TNF-α+ siRNA-TLR4 transfection group(p>0.05).2.2Results(1)The proliferation of ASMCs in TNF-αgroup was significantly higher than control group (p<0.05); The proliferation of ASMCs in TNF-α+PDTC group and TNF-α+TLR4 antibody group were significantly lower than TNF-αgroup (p<0.01); There was no significant difference in the proliferation of ASMCs between TNF-α+PDTC group and control group (p>0.05); The proliferation of ASMCs in TNF-α+TLR4 antibody group was significantly lower than control group(p<0.01).(2)The apoptosis rate of ASMCs in TNF-αgroup was significantly lower control group (p<0.01); The apoptosis rate of ASMCs in TNF-α+PDTC group and TNF-α+TLR4 antibody group were significantly higher than TNF-αgroup and control group (p<0.01).(3)The expression of NF-κB. IL-5. IL-8 protein in TNF-αgroup was significantly higher than control group and TNF-α+PDTC group and TNF-α+TLR4 antibody group (p<0.01); There was no significant difference in the expression of NF-κB. IL-5. IL-8 protein between the TNF-α+PDTC group and TNF-α+TLR4 antibody group (p>0.05);(4)The mRNA expression of TLR4 in TNF-αgroup was significantly higher than control group and TNF-α+TLR4 antibody group (p<0.01);There was no significant difference in the mRNA expression of TLR4 between the TLR4 antibody group and control group(p>0.05).(5)The protein expression of TLR4 in TNF-αgroup was significantly higher than control group and TNF-α+TLR4 antibody group (p<0.01);The protein expression of TLR4 in control group was significantly higher than TNF-α+TLR4 antibody group (p<0.01).3.Results(1)The count of EOS in airway wall asthmatic four weeks group and asthmatic eight weeks group were significantly higher than control group respectively (P<0.01). There was significant difference between the two asthmatic groups respectively (P<0.01). The count of EOS in airway wall therapeutic eight weeks group were significantly lower than those of asthmatic four weeks group, asthmatic eight weeks group and therapeutic four weeks group respectively (P<0.01). But the above-mentioned parameters were higher than control group respectively (P<0.01).(2)The airway wall thickness (WA/Pi), the bronchial smooth muscle thickness (smooth muscle area/Pi) and the number of bronchial smooth muscle nucleus(N/Pi) of asthmatic four weeks group and asthmatic eight weeks group were significantly higher than control group respectively (P<0.01). There was significant difference between the two asthmatic groups respectively (P<0.01). The above-mentioned parameters of therapeutic eight weeks group were significantly lower than those of asthmatic four weeks group, asthmatic eight weeks group and therapeutic four weeks group respectively (P<0.01). But the above-mentioned parameters were higher than control group respectively (P<0.01).(3)The airway resistance of asthmatic four weeks group and asthmatic eight weeks group were significantly higher than control group respectively(P<0.01). There was significant difference between the two asthmatic groups respectively (P<0.01). The above-mentioned parameters of therapeutic eight weeks group were significantly lower than those of asthmatic four weeks group, asthmatic eight weeks group and therapeutic four weeks group respectively (P<0.01, P<0.01, P<0.01). But the above-mentioned parameters were higher than control group respectively (P<0.01).(4)The mRNA expression of TLR4 and NF-κB in asthmatic four weeks group and asthmatic eight weeks group were significantly higher than therapeutic four weeks group and therapeutic eight weeks group and control group respectively(P<0.01); There was significant difference between the two asthmatic groups respectively (P<0.01); The mRNA expression of TLR4 and NF-κB in therapeutic eight weeks group were significantly higher than therapeutic four weeks group(P<0.05).(5)The protein expression of TLR4 in asthmatic four weeks group and asthmatic eight weeks group were significantly higher than therapeutic four weeks group and therapeutic eight weeks group and control group respectively(P<0.01); There was significant difference between the two asthmatic groups respectively (P<0.01); The protein expression of TLR4 in therapeutic eight weeks group were significantly higher than therapeutic four weeks group(P<0.05).(6)The protein expression of PCNA in asthmatic four weeks group and asthmatic eight weeks group were significantly higher than therapeutic four weeks group and therapeutic eight weeks group and control group respectively(P<0.01);The protein expression of PCNA in therapeutic eight weeks group was significantly lower than asthmatic four weeks group and asthmatic eight weeks group and therapeutic four weeks group(P<0.01). The protein expression of Bcl-2 in asthmatic four weeks group and asthmatic eight weeks group and therapeutic four weeks group were significantly higher than and control group respectively(P<0.01); The protein expression of Bcl-2 in therapeutic four weeks group was significantly higher than therapeutic eight weeks group.Conclusion1. The best time of siRNA transfected asthmatic airway smooth muscle cells is 24h.2.The TLR4 may regulate the proliferation, apoptosis and the synthesis and secretion of IL-5,IL-8 in asthmatic rat ASMCs,To participate in airway remodeling and airway inflammation in the occurrence and development. 3. The TLR4 may regulate the proliferation, apoptosis and the synthesis and secretion in asthmatic rat ASMCs by NF-ΚB transfer biological signal,playing an important role in airway remodeling and airway inflammation in asthma.4. Dexamethasone may inhibite the activity of TLR4 and the activity of NF-ΚB, then reduce the cell proliferative response and promote cell apoptosis, reduce the airway remodeling.