| 1. Background and ObjectiveMechanical ventilation is essential for the support of critically ill patients, but may aggravate lung damage, leading to ventilator-induced lung injury(VILI). The histopathological changes of VILI in lung is similar with other causes of lung injury, all accompanied by alveolar epithelium and vascular endothelium destruction, alveolar capillary membrane permeability increase, pulmonary edema and hemorrhage, hyaline membrane formation and inflammatory cells c. Since1974, webb et al., first proposed the concept of mechanical ventilation can cause lung injury, VILI is becoming more and more attention. In recent years, there are a lot of research study on the pathogenesis of VILI. Current evidence suggested that injurious mechanical ventilation with high tidal volume or high pressure could cause excessive lung tissue expansion and induce mechanical lung injury. In addition, on the basis of mechanical damage, pulmonary inflammatory cells infiltrating in lung and releasing a lot of inflammatory mediators can further exacerbate the lung injury. Depending on the type of damage, VILI is roughly divided into the following four types:barotrauma, volutrauma, pulmonary atelectasis injury, and biotrauma. Generally, the former two types appeared in the early of VILI, then biotrauma appeared in the later period, which characterized by infiltration of inflammatory cells and overproduction of pro-inflammatory mediators. The mechanism of biotrauma is very complex, have become the focus in the VILI. A large number of studies have shown that the stretch stress produced by ventilation in lung could lead to the activation of MAPK signal transduction pathway, nuclear factor-kappaB(NFκB) signaling pathway to release amount of inflammatory factors such as TNF-a, IL1-8, which cause leukocyte infiltration in lung tissues. Interaction between these inflammatory cells and cytokines, constitute a large and complex network system, not only can directly lead to lung tissue damage, can also be released into all tissues and organs through the blood circulation, cause multiple organ dysfunction (MODS) or even have life-threatening.Although in the past few decades have had a lot of research on the pathogenesis of VILI and achieved some effective results, but the exact mechanism is still understood. A recent study suggested that VILI may be related to circadian rhythm, this found provide a new mechanism for VILI. Nearly all organisms have developed mechanisms for anticipating environmental changes in order to optimize their survival. Circadian rhythms are autonomous, self-sustained,-24h oscillations in biologic processes entrained by environmental cues, most notably the daily changes in light intensity. Daily rhythms have been shown to influence peripheric organs biological processes. The lung is no exception that pulmonary ventilation function, functional residual capacity, pulmonary airway resistance have been discovered exhibit regularity oscillations, circadian clock genes expression regularly in lung is to maintain normal lung function. The nature of the processes carried out by lung and the expression of a large number of genes that regulate these processes has been suggest that may be under direct or indirect control of circadian oscillators for the efficient functioning of the organ. Furthermore, disruption of this regulation could be a cause or an effect of lung pathologies#. Research has shown that mice with chronic lag due to the abnormity of clock genes expression in the lung tissue resulting in pulmonary function changes.Nocturnal symptoms of COPD Patients accompany with sleep irregular could lead to circadian disruption also reported in previous researches. Then, as the respiratory system related diseases VILI is also with circadian rhythm? Rarely study have been reported. Therefore, this research mainly from the circadian rhythm of this new perspective to explore the pathogenesis of VILI, so as to provide a new way to prevent VILI and new drug targets for VILI.2. Materials and methods2.1Chapter one:the change of Rev-erba expression in rat lung tissue caused by mechanical ventilationThe study protocol was approved by the Institutional Animal Care Committee at the southern medical University.24Pathogen-free Sprague-Dawley rats weighing200to250g were separated into three groups according to tidal volume. All rats were maintained under standard conditions with free access to water and rodent laboratory food and maintained on a12-h light/dark (LD) cycle, artificial light was present from7A.M. to7P.M. each day, room temperature was maintained between22℃to24℃. All animals were anesthetized with10%chloral hydrate350mg/100g intraperitoneal, a catheter was inserted into the trachea under direct vision via oral cavity. Catheters were also inserted into the femoral artery and vein. Femoral artery catheter to collect biological signals, continuous monitoring mean arterial pressure (MAP) and heart rate (HR). Lactated Ringer’s solution was infused at a rate of10ml/kg/h through the venous catheter. During the experiments, the rats were placed under a warming light and rectal temperature was maintained close to37℃. The control group (n=8) underwent spontaneous breath. A second group (LV group)(n=8) was mechanically ventilated with10ml/kg tidal volume MV for2h, whereas rats in the third group(HV group)(n=8) mechanically ventilated with40ml/kg tidal volume for2h. During mechanically ventilated rats were paralyzed by a continuous infusion of0.1ml/kg/h vecuronium bromide. The respiratory rate was set to40times per minute for the LV group,80for the HV group. Rats were released to the cage and kept separately from each other for24hours after the treatment was finished.24hours after the successful establishment of animal model, all rats were painless killed by chloral hydrate anesthesia and whole lung tissues were isolated from thoracic cavity. All of the left low lobe were collected to measure the Wet-to-dry Ratios, and the left upper lobe of lungs for histopathologic examination. The others were stored at-70℃until the protein and RNA assays were performed.2.2Chapter two:the treatment effect of SR9009on ventilator-induced lung injuryWe assigned another16rats into two groups of8animals treatment with SR9009or not. The treatment group (name as SR9009) was injected50mg/kg SR9009before anesthesia, while the other group (non-SR9009) was injected the same amount of normal saline (NS) replace.30min later, all animals were anesthetized with10%chloral hydrate350mg/100g intraperitoneal, a catheter was inserted into the trachea under direct vision via oral cavity. Catheters were also inserted into the femoral artery and vein. Femoral artery catheter to collect biological signals, continuous monitoring mean arterial pressure (MAP) and heart rate (HR). Lactated Ringer’s solution was infused at a rate of10ml/kg/h through the venous catheter. During the experiments, the rats were placed under a warming light and rectal temperature was maintained close to37℃. Both groups were suffered40ml/kg tidal volume mechanically ventilation for2hours, the respiratory rate was set to40times per minute. During the mechanically ventilated rats were paralyzed by a continuous infusion of0.1ml/kg/h vecuronium bromide. Two hours later, all rats were free separately for24hours.24hours after the successful establishment of animal model, all rats were painless killed by chloral hydrate anesthesia and whole lung tissues were isolated from thoracic cavity. All of the left low lobe were collected to measure the Wet-to-dry Ratios, the left upper lobe of lungs for histopathologic examination, and the others for Elisa analysis.3. Results3.1Pulmonary Histopathologic Changes and wet/dry ratioMV led to pulmonary inflammation and injury, as indicated by thickening of the alveolar septum and infiltration of inflammatory cells, evident in histopathological examination of the lung samples taken in the groups of LV, HV and non-SR9009. In the presence of SR9009, both edema and inflammatory cell infiltration were reduced. W/D ratio is the evaluation index of pulmonary edema. Compared to the control group and the low tidal volume group, the W/D value of high tide volume group increased significantly (P<0.05), suggest that the group after2h of high tide volume MV causes serious lung damage and pulmonary edema.3.2Clock genes expression in lung homogenatesCompared to breathe freely group, bmall and clock genes mRNA increased slightly, but Per2gene mRNA slightly decreased in high tide volum MV group with no significant difference. While Rev-erb alpha gene mRNA was significantly decreased (P<0.05), the same as REV-ERBa protein product which performed by western blot approach in high tide volume MV group compared with control group. These results suggest that REV-ERBa may piay an important role in VILI.3.3TNF-a levels in lung homogenates treated by SR9009 Compared to breathe freely group, the concentration of TNF-a in high tide volume group significantly increased,P<0.001.Because TNF-a is a key pro-inflammatory in VILI, we investigated the effects of REV-ERBa synthetic ligand SR9009treatment during high tide volum MV on the expression level of TNF-a. ELISA(enzyme linked immunosorbent assay) demonstrated that SR9009significantly reduced VILI-induced expression of the pro-inflammatory cytokine TNF-a.These results suggest that the ability of REV-ERBa to prevent VILI can be regulated by its synthetic ligands.4. Conclusions(1) Injurious mechanical ventilation with high tidal volume or high pressure could cause an increase in microvascular permeability, lung edema and hyaline membrane formation, which is the major pathological changes of VILI. this study successfully built the VILI animal model;(2) The rat lung injury caused by mechanical ventilation may be occured circadian rhythm disorder; Rev-erba plays an important role in protection rats lung from mechanical ventilation damage;(3) Rev-erb alpha protein specific agonists SR9009has a good therapeutic effect on VILI, can significantly reduce the degree of lung injury and decrease the TNF-a level in rats caused by high tide volume mechanical ventilation so that alleviate the inflammation response in lung. |
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