Experimental Studies On The Change Of Circadian Rhythm And Gene Expression In Lung Of Ventilation-induced Lung Injury In Rat

Backgroud and ObjectivesVentilation-induced lung injury is a kind of injury by releasing cell factors to the most alveolar-capillary membrane and its incidence accounts for about 5% to 39% in mechanical ventilation. The pathogenesis of VILI is complicated, related to traditional barotrauma, volutrauma, pulmonary atelectasis injury, and biological injury, but so far has not yet fully understood. Recently many researchers reported that respiratory diseases of lung cancer, chronic obstructive pulmonary disease, asthma and ischemic brain injury are caused by breaking circadian rhythms, and used chronotherapy can achieve effective function. As the acute injury of respiratory system, VILI may exist breaking circadian rhythm as well. VILI is part of the systemic inflammation, or specific response against the lung tissue .It caused by a variety of cells and factors and with expression of multiple genes and pathways. Hence, the study of these genes and pathways is very important to clarify the pathogenesis of VILI. A lot of genes about VILI had been discovered, but we still need detect the level of whole-genome, in order to comprehensively research on VILI. Gene chip as a kind of high-throughput screening and efficient tool is an effective method of studying gene expression. This study was used Affymetrix whole genome expression microarray technology, to screen differentially expressed genes in rats of VILI and analyze the function of these genes in order to find the circadian rhythm and further explore the molecular mechanism of VILI.Methods1 Preparation the model and discussion of the circadian rhythm of VILI72 Sprague Dawley rats were adaptive bred for 2 weeks, and randomly divided into four groups at 4 time points (1AM, 7AM, 1PM and 7PM) each for 18. Then the rats in groups at each time point were randomly subdivided into three groups again. Group FB (n=6): free breathing group, with non-ventilation; Group LV (n=6): with low Vt ventilation (Vt=8ml/kg, PEEP=0); Group MV (n=6): with high Vt and no PEEP (Vt=40ml/kg, PEEP=0). The parameters of arterial pressure, and heart rate, blood gas changes of rats were monitored. After ventilation for 3h, the rats were killed by exsanguination and the samples were obtained. The changes of histopathology with H&E staining and the ultramicrostructure of the pulmonary cells were also observed by light microscope. And liquid chip technology was used to test the parameters of plasma corticotropin hormone (ACTH) and Meltonin (MT) level.2 Screen rat differentially expressed genes of VILI by Gene chip technologyThe male rats from 1AM and 1PM time point of Group FB and Group HV were selected. Then RNA from each rat was extracted and mixed by different time point and group. After preparation, Affymetrix Rat Genome 230 2.0 Array was used to test the whole-genome of rat. Hence, RT-PCR technology was used to test Cyp1a1,Npas2,Nr1d1 genes of 72 rats in order to validate the dependability of the gene chip technology , and to find the probable mechanism of VILI.3 Statistic analysisThe experimental results are showed by mean±standard deviation((x|-)±s). Datas were analysed by SAS 9.1. When P<0.05, it has statistically differences. When P<0.01, it has statistically significant differences. After test of normal distribution and homogeneity of variance, datas were analysed by one way anova, and Turkey test when interclass paired comparison approach was needed. The cosine rhythms function, F(t)=M+A cos(ωt+Φ) was structured to analyse rhythm. When P<0.05, it has statistically differences and rhythmicity. The higher the ratio of A/M, the stronger the rhythmicity.(A stands for Amplitude, M stands for mesor.)Results1. There are no significant differences in the parameters of arterial blood pressure and heart rate, Meltonin. They were no rhythmicity by cosine analysis. The values of ACTH had statistically significant differences.(P<0.01)2. Arterial gas analysis: The basic values of pH,and PaCO₂,PaO₂ were no statistically differences. After mechanical ventilation for 3h, compared the basic values, the values of PaCO₂ were statistically differences with Group HV, P<0.05.3. Score of lung injury: Group HV had statistically significant differences with Group FB and Group LV.(P<0.01)4. Altered gene expression in VILI. Compared Group HV with Group FB by gene chip, there were 56 differential expressed genes, among which 43 up-regulated and 13 down-regulated. Il-6,Cox-2 and Ccl2 genes were up-regulated. Different gene function was classified as follows: a. inflammation relevant genes, b. metabolism relevant genes, c.immunization relevant genes, d. signal transduction relevant genes, e. others such as oxidation and antioxidation relevant genes.5. The results of Cyp1a1, Npas2, Nr1d1 genes detected by RT-PCR accorded to the results by gene chip. Comparing with Group HV, Gene Nr1d1 was statistically differences, P<0.05. Cosine analysis showed Nr1d1 had rhythmicity in Group FB.Conclusions1. These results suggested that the high tidal volume ventilation could cause the pulmonary structure destroy, lung edema and also cause the severe inflammatory response into lungs. That is to say the model of VILI is successful.2. There were no differences between free breathing and mechanical ventilation in the plasma level of Meltonin. So were the circadian rhythm. But there were changes in the plasma level of ACTH.3. The molecular mechanism of VILI is very complex, maybe the synergy of multiple genes .It relates to signaling pathways, oxidatie stress reaction, metabolic pathway, immune response and other kinds of change.4. The pathways of VILI were so many. Therein to,IL-6 and chemotactic factors may be the canal commune of VILI.5. Down expression of gene Nr1d1 may lead to over expression of IL-6 and Cox-2, thus induce NK-kappa B active, and then cause acute lung injury.