The Effect And Mechanism Of The Secretion By Different Dose Of Lipopolysaccharide In Asthmatic Rat ASMCs

Objective:We will establish asthmatic rat model,study the different dose of Lipopolysaccharide (LPS) to stimulate asthmatic rat airway smooth muscle cells(ASMCs) and to activate Toll-like receptor4from cell level animal experiment, comprehend the effect of the synthesis and secretion mechanism in asthmatic rat ASMCs.Methods:Using OVA to Sensitize and challenge to established asthmatic rat model,separation of ASMCs and culture ASMCs,by using different dose of LPS to stimulate asthmatic rat ASMCs and TLR4antibody to block. Measuring the concentration of cytokine IFN-γ、IL-4in supernatant of asthmatic rat ASMCs by ELISA.The expression of TLR4mRNA in asthmatic rat ASMCs was analyzed by quantitative real-time(RT-PCR).And to study the secretion of cytokine by using the different dose of LPS to stimulate ASMCs cultured in vitro the expression of TLR4mRNA.And we also established asthmatic rat model, by using different dose of LPS to inject the rats,and to observe the histopathology change of rat pulmonary tissues by hematoxylin and eosin(HE),to analyzed the expression of TLR4mRNA in asthmatic rat airway smooth muscle by RT-PCR,to investigate the effect of asthmatic rat airway smooth muscle by different dose of LPS and the expression of TLR4mRNA.1.Results(part I)(1) Measuring the concentration of cytokine IFN-γ、IL-4in supernatant of asthmatic rat ASMCs by ELISA:The expression of IL-4in supernatant in low dose of LPS group detected significantly higher than control group (P<0.01) the expression of IFN-γ in supernatant and control group showed no significant differences (P>0.05). The expression of IL-4in supernatant in high dose of LPS group showed significantly lower than control group (P<0.01),the expression of IFN-y in supernatant detected significantly higher than control group (P<0.01). The expression of IL-4in supernatant in low dose of LPS group detected significantly higher than high dose of LPS group (P<0.01), the expression of IFN-γ in supernatant detected significantly lower than high dose of LPS group (P<0.01).The expression of IL-4in supernatant in low dose of LPS+TLR4antibody group detected significantly lower than low dose of LPS group (P<0.01), the expression of IFN-γ in supernatant and low dose of LPS group showed no significant differences (P>0.05),the expression of IL-4and IFN-y in supernatant and control group showed no significant differences (P>0.05).The expression of IL-4in supernatant in high dose of LPS+TLR4antibody group detected significantly higher than high dose of LPS group (P<0.01),the expression of IFN-γ in supernatant detected significantly lower than high dose of LPS group (P<0.01), the expression of IL-4and IFN-γ in supernatant and control group showed no significant differences (P>0.05)(2) The expression of TLR4mRNA in asthmatic rat ASMCs was analyzed by RT-PCR:The expression of TLR4mRNA we analyzed in low dose of LPS group and high dose of LPS group significantly higher than control group (P<0.01),and high dose of LPS group significantly higher than low dose of LPS group (P<0.05).The expression of TLR4mRNA we detected in low dose of LPS+TLR4antibody group and high dose of LPS+TLR4antibody group significantly higher than control group (P<0.01). The expression of TLR4mRNA showed in low dose of LPS+TLR4antibody group significantly lower than low dose of LPS group (P<0.01).The expression of TLR4mRNA showed in high dose of LPS+TLR4antibody group significantly lower than high dose of LPS group (P<0.01)2.Results(partⅡ)(1) HE in lung tissue:asthma group showed there are lots of inflammatory cells infiltration round the bronchi, pulmonary alveoli and blood vessels.The wall of bronchus thickening, mucous membrane swelling and hyperemia,wrinkle increased, airway smooth muscle thickening and lumens stenosis. There were no significantly inflammatory decreased changes in low dose of LPS group.But above-mentioned changes in high dose of LPS group showed significantly decreased changes.However, we observed there were no significantly inflammation and normal lung tissue in control group.(2) The count of EOS of airway wall in asthma group,low dose of LPS group and high dose of LPS group were significantly higher than control group (P<0.01). The count of EOS of airway wall in low dose of LPS group were significantly higher than asthma group (P<0.05).But the count of EOS of airway wall in high dose of LPS group were lower than control group (P<0.05).The count of EOS of airway wall in low dose of LPS group higher than the high dose of LPS group(P<0.01).(3)The bronchi wall thickness (WAt/Pi), the bronchial smooth muscle thickness (WAm/Pi) and the number of bronchial smooth muscle nucleus(N/Pi) of the asthma group,low dose of LPS group and high dose of LPS group analyzed significantly higher than the control group(P<0.01). The above-mentioned parameters of low dose of LPS group showed significantly higher than asthma group(P<0.01).The above-mentioned parameters of high dose of LPS group showed significantly lower than asthma group(P<0.01).(4)Airway resistance:we found that the airway resistance in asthma group,low dose of LPS group and high dose of LPS group showed significantly higher than control group (P<0.01).The airway resistance in high dose of LPS group detected significantly lower than asthma group (P<0.01). There was no significant difference in airway resistance between low dose of LPS group and asthma group (P>0.05).The airway resistance in high dose of LPS group detected significantly lower than low dose of LPS group (P<0.01)(5)The expression of TLR4mRNA in asthmatic rat airway smooth muscle by RT-PCR:the expression of TLR4mRNA we detected in asthma group,low dose of LPS group and high dose of LPS group significantly higher than control group (P<0.01). The expression of TLR4mRNA we showed in low dose of LPS group and high dose of LPS group significantly higher than asthma group (P<0.01). The expression of TLR4mRNA we analyzed in high dose of LPS group significantly higher than low dose of LPS group (P<0.05)Conclusions:(1) LPS may activate TLR4in ASMCs of asthmatic rat and make ASMCs secretion Th1/Th2imbalance, accordingly may involve in airway inflammation of asthma.(2) TLR4plays an important role in asthmatic airway inflammation and airway remodeling, LPS may play double-sided regulation in asthmatic airway inflammation and airway remodeling by activated TLR4.