Effects Of Carbon Monoxide On LPS-induced Damage And Possible Mechanisms In Rat Alveolar Macrophages NR8383

Acute respiratory distress syndrome(ARDS) caused by complex etiology is a clinical common critical disease, which develop rapid and have high mortality. The alveolar macrophages, as the main residence of phagocytes, constitute the lung respiratory pathogen's first line of defense, which play an important role in the occurrence, development and outcome in ARDS. When toxic substances or sensitized particles entered respiratory tract, alveolar macrophages can be activated as well as neutrophils. Activated cells producing a large number of oxygen free radicals and secretion of pro-inflammatory factor may lead to cell apoptosis and lung damage. Studies have shown that mitochondrial is main venue of reactive oxygen species(ROS). Mitochondria suffer oxidant stress which contain normal oxidative phosphorylation pathway inhibition and decreased the activity of endogenous free radical scavenger resulted in a large number of ROS generated, mitochondrial membrane potential decreased and ATP production decreased. The above mitochondrial changes cause cell oxidative stress damage in ARDS. Mitochondrial function is closely related to the morphological structure. when cells suffer oxidative stress damage the balance between mitochondria fusion and fission is broken, that is fission more, fusion less.Carbon monoxide(CO) is known as a kind of pollution and harmful gas in the atmosphere. Claude Bernard recorded CO can be combined with hemoglobin, which could weaken even deprive the binding force of O2 and hemoglobin eventually lead to asphyxia in 1857. However, researches have found that CO played an important role in many pathological state in animal models. The researches is mainly around the CO ability of anti-inflammatory role, resistance to oxidative stress and apoptosis and cell protection ways in recent years. These functions are all under stress state in cells. Studies have demonstrated that CO can reduce oxidative stress caused by isoflurane in rat brain cells through the regulation of mitochondrial cytochrome oxidase activity. whether the carbon monoxide play a role against LPS caused alveolar macrophages damage by influencing the morphology and function of mitochondria remain to be studied.Objective To explore effects of CO on LPS induced damage in rat alveolar macrophage NR8383 and possible mechanisms.Methods This study was divided into two parts. Part 1: Different concentrations of LPS and CO releasing molecule-2(CORM-2) were added into NR8383 to determine the concentration of LPS induced NR8383 damage model and CORM-2 working concentration. Part 2: Cells were randomly divided into 8 groups(n=10).Treatments of every group were as follows: CORM-2 100?M was added into CORM-2 group. ZnPP 10?M was added into ZnPP group. LPS 10?/ml was into LPS group. LPS+CORM-2 group cells were pretreated with CORM-2 100?M for 1h, then cultured with LPS 10?/ml. LPS+iCORM-2 group treatments was same with LPS+CORM-2 group except for replacing CORM-2 with iCORM-2. LPS+ZnPP group cells were pretreated with ZnPP 10?M for 0.5h, then cultured with LPS10?/ml. LPS+NaOH group treatments was same with LPS+ ZnPP group except for replacing ZnPP with NaOH. The amount of PBS was added into group C. Cell precipitation and supernatant was collected after treatments 24 h for targets detection. Proliferation was measured by MTT assay, ROS was detected by DCFH- DA probe, SOD activity was tested by SOD kit, apoptosis and mitochondrial membrane potential were detected with flow cytometer, content of ATP was tested by ATP content kit, HO-1, Drp1, Fis1, Mnf1/2 and OPA1 mRNA was measured by RT-PCR, HO-1, Drp1, Fis1, Mnf1/2 and OPA1 expression was determined by Western blot.Results Part1: LPS induced NR8383 damage model was successfully built with LPS concentration 10?/ml. CORM-2 safe working concentration was 100?M. Part2: LPS induced NR8383 damage, however this damage condition can be alleviated by pretreatment CO. Furthermore, cytoprotection of CO is associated with HO-1 to regulate mitochondrial fusion and fission protein expression and function.Conclusion CO can alleviate LPS-induced damage in rat alveolar macrophages through HO-1 to regulate mitochondrial fusion and fission protein expression and function.