Study On Inflammatory And Immune-related Gene Expression Profile And The Effect Of RhoA Gene On Regulation In ARDS Rats

Objective:The aim of this study was to investigate the inflammatory and immune related genes’expression of acute respiratory distress syndrome (ARDS) in lung tissue of rats. We intervene in ARDS rats with AG490and GCs’s to observe RhoA gene expression changes. At the same time, considering the macrophage’s important role in inflammation and ARDS, we examined the related genes’expression in the macrophagic system RAW264.7which is induced by LPS and intervened by SiRNA, AG490and GCs simultaneously. Then, investigate the mechanism of ARDS from the related gene’s expression variation and RhoA gene’s regulationiMethods:1. ARDS rats model was established by sublingual injection of oleic acid.2. The inflammatory and immune-related genes of lung tissues of ARDS rats were tested by genechip technique, and quantitative fluorescence RT-PCR was used to validate the results.3. The expression of RhoA mRNA and LIF was investigated by real-time PCR and ELISA respectively.4. Treat RAW264.7macrophages with LPS(1μg/ml),and at the time points:2h,6h,12h,24h, examine the expression of RhoA mRNA by Real time-PCR, then intervene these cells with SiRNA, AG490and GCs, detect the expression of RhoA by RT-PCR and the level IL-6by ELISA respectively at6h,12h,24h time points.Results:1. Oleic acid induced ARDS rats model were established and validated by blood gas analysis and pathological observations.2. The genechips’ results showed that4genes were up-regulated (RhoA, Lif, Serpine1and Tac1)and10genes were down-regulated(RT1-T24-1, RT1-S3, RT1-149, Cfh, Casp12, Hspa8, Scin, Cyp2e1, Cyp2b3,and Itpka) with1.5times in lung tissues of ARDS rats, which associated with inflammatory and immune response and cytoskeleton regulation. The results of quantitative fluorescence RT-PCR bear out the data of genechips.3. The results of AG490and GCs intervention in ARDS rats showed that the histopathological situation in lung of ARDS group is the most serious, AG490and GCs group are better, and that of the control group is normal entirely.The results of Real time-PCR showed that the expression of RhoA mRNA in lung tissues of ARDS group was the highest, then were AG490group and GCs group, the control group was the lowest. LIF expression variation was similar to that of RhoA.4. After treated RAW264.7macrophages with LPS, the results of Real time-PCR showed that the level of RhoA mRNA was no significant difference at2hours,24hours between LPS group and control group, however at6and12hours RhoA mRNA expression of LPS group was higher than that of the control group.5. After intervened with RNAi, AG490and GCs, RhoA mRNA expression levels were significantly down-regulated compared to LPS group, while that of SiRNA-1group at12and24h was much lower than the normal group; RhoA mRNA levels of LPS group reached the highest at6h,then declined to normal at24h. The level of IL-6of LPS group were the highest at all different time points, and that of SiRNA group, AG490and GCs group were lower than that of LPS group,but higher than that of the control group.