The Relativity Research Of 5-lipoxygenase And Lung Injury Caused By Panaquat Poisoning

ObjectiveParaquat(PQ) used as weed killer was widely used by over 130 countries,Since it went on the market in 1962.The cases causing death by PQ was firstly reported in 1966.PQ poisoning can cause multiple organs injury,lung is the most important injuried target organ.Acute alveolar catarrh and pulmonary interstitial fibrosis lastly growing are the major reasons which caused by PQ poisoning.To lessen the injury of lung is the major therapia of PQ poisoning.The mechanism of PQ poisoning has not yet been elucidated.But a great quantity studies showing:inflammatory cells and multiple cytokines like poltmor phonuclear neutrophils(PMN) play an important role in inflammatory reaction.Cytokine network runaway has been gradually became the focus which are paying close attention by exterior and inferior researchers.How to control the excess releasing of inflammatory cytokines,and to balance the cyrokine network,which has became hot spot to reseach.Leukotrienes(LTs) are lipid mediators of inflammation derived from the 5-lipoxygenase(5-LO) pathways of the arachidonic acid metabolism.IL-13 is a cyrokine that is produced in large quantities by lung alveolus macrophage and neutrophil.LTs stimulus the marrow multifunction haemopoietic stem cells and these cells transmigrate in the blood that affect the function of White corpuscle.LTs also can enhance the expression of the adhesion molecules,promote cells movement and shift to the tissue.Once White corpuscle reach to inflammatory tissues,LTs can enhance its viability and activity.LTs mainly mediate the recruitment of neutrophil,mononuclear macrophage and labrocyte.Leucocytic chemotaxis causing by LTs in lung inflammatory reaction may elicit the water fall effect of lung inflammatory reaction.Baud's studies demonstrated that LTs is the 5-LO downstream production which play a immediately role in fibrocyte's migration and generation,also accerementition of the matrix protein.Exogenous LTD4 lead to murine's and human's fibrocyte reduplication which submit to dose dependent.Studies showing that LTS as an important mediators of inflammation play an significant role in pathogenesy of many inflammatory diseases,which has been gradually recognized by people.But its action in PQ poisoning has not yet been clear.The study aim to approach the relativity of 5-LO and lung injury caused by panaquat(PQ) poisoning from the angle of the inflammatory cytokine network.Materials and Methods1,Animal subgroupA total of 40 wistar rats were randomly divided into control group and poisoned group.Intra-gastric administration with PQ in dose of 50mg/kg and normal saline in the same dose were given the control group.On the day of 1,3,7,14 after exposure ten of them was put to death,and then collect the bronchoalveolar lavage fluid and Raji cell assay Raji,the level of 5-1o and mRNA in lung tissue were observed by RT-PCR and immunohistochemistry.2,Bronchoal veolar lavage fluid(BALF) collection and Raji cell assay RajiUse in vivo whole lung lavage to collect 5ml BALF.1500rpm,4℃centrifuging for 10 minutes,and put centrifugated cell aggregate suspending with 0.3ml normal saline,then fiat cell suspension.Total BAL White Blood Cells were enumerated by counting on a haemocyrometer.3,RNA extraction and RT-PCRExtract the total RNA with TRIZOL,inverse transcription as CDNA. 5'-CTCCCAGTGACCACAGAA-3'of the 5-LO gene upstreen primer,downstream primer is 5'-ATACCGAACACCTCAGACA-3',amplified fragment length was 496bp,β-actin as infernal reference(amplified fragment 247bp),circulation conditions: 95℃,degeneratefor5minutes,94℃for30 seconds,-54℃lminute,-72℃30 seconds,all for 35 circulations.72℃extending for 5 minutes with 15%of gelose gel electrophoresis.4,ImmunohistochemistryDetect the expression of 5-LO with pulmonary Paraffin section immunohistochemistry,Streptavidin-Peroxidase which follow the kits instruction.Result1,Pathological changeObserving the cut sheets with HE stain after exposure for 1 day,the structure of lung alveolus can be clearly visible on the third day.Small amounts of inflammatory cells leaked out in alveolar space.On the seventh day,the structure of lung alveolar acceptable recognized,alveolar space was filled with massive inflammatory cells and the diffusate began to organiz.The structure of alveolus was not clear to the fourteenth day,there was full of inflammatory cells,but the total numbers of cells were decreaced comparing to before,fibrous degeneration can be seen.2,The changing of numbers of white blood cells in BALFThe total number WBC in poisoned group compared to control group had statistical significance(p＜0.01).The level of WBC began to increase on the first day after poisoning and reached to the peak on the seventh day.On the fourteenth day the level was decreased comparing to the seventh day.3,The changing of 5-LOComparing to the control group,the expression of 5-LO in poisoned group have statistical significance.(p＜0.01) The level of 5-LO began to increase on the first day and reached the peak on the seventh day.Comparing to the seventh day,the level was decreased on the fourteenth day.The changing of the protein level is the same as genes. ConclusionThe changings of the WBC total number and the tendency of the changing of pathology are with one accord on the whole.The tendency of the changing of 5-LO protein level is the same as the genes.The changing of WBC total number in BALF comparing to the IOD values of the result of 5-LO immunohistochemistry have significant positive correlation.(r=0.981 p<0.001) Our studies demonstrate that 5-LO play an important role in PQ induced acute lung injury and fibrosis by affect the numbers of WBC in lung.