Endotoxin-induced Lung And Kidney Injury And Cilostazol Attenuates Lung And Kidney Stress Response

Sepsis is one of the high mortality in clinical diseases, especially in thedepartment of respiration and ICU. There are many kinds of bacteria, most ofE.coli, that can cause sepsis and high in clinics. The main reasons of sepsisresulting into mortality are acute multiple organ function damage andmultiple organ failure. To explore the pathogenesis and treatment of sepsis isstill a hot issue of research to find effective medicines for sepsis. They allwant to develop a new method to treat sepsis.In acute inflammation, the inflammatory cells include neutrophils,monocytes, macrophages and so on. Some inflammatory factors are common,such as prostaglandin, histamine, leukotriene, platelet activating factors,interleukin and tumor necrosis factor et al. Vascular reaction is considered tobe the central key of the inflammatory process. In sepsis, actived neutrophilsand other inflammatory cells induced a variety of inflammatory factors andadhesion molecules. Increased vascular permeability and leukocyteinfiltration can cause tissue edema and inflammation.The main inflammatory pathways caused by LPS are NF-kB pathway, MAPK/ERK pathway and so on. NF-kB, the main medium and transcriptionfactor in the inflammatory reaction, can combine specifically with a varietyof gene promoter or enhancer with specific sites to promote genetranscription. Sustained activation of MAPK may lead to producinginflammatory cytokines continuously and persistent inflammation andincreasing damages, called waterfall reaction. During the above process,p38MAPK was more important signal. It had a close relationship withNF-kB pathway and was a main factor of the activation of NF-kB pathway.The activation of NF-kB pathway and p38MAPK pathway can lead toinflammatory cytokine cascade waterfall reaction and the increase of oxygenfree radicals, which resulted into cell damage and death. Therefore, if theactivation of most of inflammatory pathways was blocked, we can get bettertreatment results.Cilostazol is a medicine for anti-platelet aggregation, which is foundplaying an important role in reducing inflammation in the recent experiments.In order to study the progression of inflammation of LPS induced sepsis, andthe mechanism of cilostazol reduces the stress of LPS on lung and kidney,we used C57mice and made the sepsis model with LPS by intraperitonealinjection. We also gave cilostazol by intraperitoneal injection and usedpathology and immunofluorescence staining, ELISA and Western blot to testthe samples.The first part of experiment: The study about the injury of renal and lungcaused by intraperitoneal injection of LPS. Through intraperitoneal injectionof endotoxin (LPS), the animal model of sepsis was set up successfully.Based on different purposes of the study, we made two kinds of models: Firstone was made to observe the pathological changes. The models mice were killed24hours after intraperitoneal injection of LPS (5mg/Kg) and theirtissues were kept as samples to observe the effects of endotoxin on thestructure of lung and kidney tissue by pathological staining in renal and lungtissue and immunofluorescence staining in frozen slice; Second one wasmade to study the variation of some factors stimulating inflammatory factors.The mice were killed6h and24h, respectively, after intraperitoneal injectionof LPS (7.5mg/Kg). The blood, lung and renal tissues from mice werecollected to determine the level of MPO in lung tissue, levels of BUN inserum and inflammatory factor in kidney, which can evaluate the extent oforgans damage. The levels of proinflammatory cytokines IL-6and TNF-alpha were determined by ELISA. And the activation of related inflammatoryfactors (ICAM-1and VCAM-1) and inflammatory pathway of ERK and thechanges of Mst-1were determined by Western Blot.Through the experiment we found that the main pathologic change. Theinfiltration of inflammatory cells, was observed in early stage of sepsiscaused by endotoxin (LPS). These inflammatory cells infiltrated to theorganization through the vascular wall, but the destruction on the overallorganization structure was not obvious. Evaluation of lung and renal damage:the levels of MPO in lung tissues after6hours and24hours when sepsiswere significantly increased, but there was no significant difference betweenthe two time points. The levels of BUN in serum increased significantly withtime going. The levels of IL-6and TNF-α were significantly high: nosignificant statistical differences of the increase of IL-6were found between6h and24h, while the level of TNF-α after6hours was higher than that after24hours. Mst-1significantly decreased by time.The second part of experiment: Study of cilostazol reducing kidney and lung injury. In order to clarify the mechanisms of reducing inflammation, weuse of cilostazol in treatment of sepsis. We gave LPS (5mg/Kg) byintraperitoneal injection to make the model. For the treatment group, the micewas given cilostazol0.3mg/mouse by intraperitoneal injection. In order toreduce the interference of cilostazol solvent, PBS group and LPS group wereinjected with the same volume of DMSO at1hours after LPS injection. After24hours of the first injection, blood、lung tissues and kidney tissues of micewere collected. In order to discover the change in the levels ofproinflammatory factor TNF-alpha, we use the ELISA method. To find thechanges effects of cilostazol on inflammatory factors in the role of VCAM-1,p-Erk, p-p38, we used Western Blot to find the activation of inflammatorypathway, such as ERK/MAPK pathway and p-p38pathway in kidney; inorder to understand the effect of cilostazol on oxidative stress, MnSOD,HO-1, and TBARS were also measured. We also use Western Blot andELISA method to exam the lung tissues.In our experimental results, between the treatment group (LPS/CSZgroup) and the model group (LPS group), the levels of TNF-α in the serumof mice decreased significantly. The treatment group compared with themodel group, we also found the debasement of HO-1, the TBARS testdecreased and the level of MnSOD than in the LPS group. The above datademonstrated that cilostazol has anti-oxidative stress effect. Reduced theactivation of p-ERK and p-p38signaling pathway made inflammationdecreased. Experiments show that, cilostazol has a protective effect onendotoxin induced renal injury, but the protective effect on the lung tissuewas not obviously.