Analysis Of Prognostic Factors Of Paraquat Poisoning And Protection Mechanism Of Dexamethasone On Paraquat-Induced Cytotoxicity Of Pulmonary Epithelial Cell In Mice

ObjectiveParaquat?Paraquat,PQ?is a kind of quick contact quaternary ammonium salt steriland herbicide,which is widely used in agricultural production and caused varies damage absorbed by digestive tract,respiratory tract,skin integrity and other ways.Because of the special effects of alveolar type I epithelial cells and alveolar type II epithelial cells on their active uptake and accumulation,PQ can accumulate abnormally high concentration in lung tissue.The concentration of PQ in lung tissue is 6-10 times higher than that in plasma,leading to acute respiratory distress syndrome,rapidly the development of acute respiratory failure or serious damage to permanent lung.After poisoning,the condition is critical,and the fatality rate is extremely high.Even the surviving patients suffer from severe pulmonary fibrosis that affects the quality of life and work capacity of the patient.At present,the mechanism of paraquat induced acute respiratory distress syndrome is not clear,and there is still no treatment effect,and the mortality rate is extremely high.In this paper,the clinical data of PQ poisoning were analyzed and summarized.The factors affecting the prognosis of PQ poisoning patients were analyzed and discussed,which deepened the understanding of the clinical characteristics of PQ poisoning.At the same time,experimental study on the cellular level,to explore the mechanism of hormone induced lung epithelial cells against PQ injury in mice induced by PQ in mice and to clear the mechanism of lung epithelial cell injury and the effect of hormone in mice lung epithelial cell injury in the process,in order to get a better treatment plan,reduce the mortality rate and improve the quality of life of patients.MethodPart one172 medical records of acute paraquat poisoning patients were collected from the poisoning emergency center of the Affiliated Hospital of the armed police Logistics College from January 1,2010 to May 30,2016.1.The general situation,clinical manifestations,treatment plan of patients were collated,follow-up the prognosis of patients.2.Collect the blood and urine concentration of PQ on admission,blood SIPP,urine SIPP,complete blood count,liver function,renal function,electrolytes and blood gas analysis.The differences of these indexes between the death group and the survival group were compared.3.Important clinical indicators of PICCO index were collected?for example,extravascular lung water index,pulmonary vascular permeability index,intrathoracic blood volume index?to explore its prognostic value.4.Lung function on the fifth days were collected after PQ poisoning,including VT,FVC,,FEV₁,FEV₁/FVC,RV,TLC,RV%/TLC,TLCO,to investigate value of lung function in judging the prognosis of patients with paraquat poisoning.Second partTo explore the role of TLR4 signaling pathway in the protection of PQ induced pulmonary epithelial cells in mice.Mouse lung epithelial cell lines?MLE-12?are selected as the object to be studied.Firstly,MTT is used to detect the influence of PQ on the proliferation of mouse lung epithelial cell lines,with PQ concentration in IC50being determined.Then,PQ induced cells are confirmed,after which,detection is done for the influence of DXM on the proliferation of mouse lung epithelial cell lines induced by PQ.According to the above two experiment results,the follow-up experiment is done with the groups being divided as blank control group,DXM control group,PQ group,the treatment group of PQ with low dose of dexamethasone,the treatment group of PQ with medium dose of dexamethasone,and the treatment group of PQ with high dose of dexamethasone.The influence on morphology of MLE-12 is observed in the above groups.SOD,MDA and HYP expression levels in the supernate being detected using the differential spectrophotometric method.ELISA is used to detect the contents of TNF-?and IL-6 in the supernate,RT-qPCR is used to detect the mRNA levels of TLR4,NF-?B1,TNF-?,and IL-6,and Western Blot is adopted to detect the protein expression levels of TLR4,I?B?,p-I?B??NF-?B1?p-NF-?B1?TNF-?,and IL-6.Immunofluorescence assay is done to detect the nuclear entry of NF-?B1,with NF-?B1 luciferase reporter assay being conducted to detect the activation of NF-?B1 as well as the expression of downstream genes.ResultPart oneA total of 172 patients with complete information were collected,including male:female?100:72?,with an average age of 40.4±5.8.There were 94 cases with liver injury and 119 cases with kidney injury.The average poisoning amount was 73.7±15.6ml,the poisoning time was 10.67±6.08 hours,the blood PQ concentration was6.28±3.25mg/l,the urinary PQ concentration was 15.75±5.32mg/l,the blood SIPP was 67.05+18.98,and the urinary SIPP was 168.05+34.18.Comparison of death group and survival group related laboratory indicators show that oral dose of PQ,the concentration of serum PQ and urine PQ concentration,blood SIPP,urine SIPP,white blood cell count?WBC?,Lac and PQ blood clearance time has significant difference in both groups.86 cases were monitored by PICCO,PVPI?12h?in death group is higher than the survival group,EVLWI?24h?in death group is higher than the survival group.EVLWI?24h?and the concentration of serum PQ,blood SIPP is the independent prognostic factor for paraquat poisoning.79 cases of pulmonary function test were performed on the fifth day after admission.FVC,TLC and TLCO in death group were lower than those in death group,and RV%TLC in death group was higher than that in survival group.Logistic regression analysis showed that RV%TLC was an independent predictor of prognosis in patients with paraquat poisoning.Follow up:109 patients died and 63 survived,with a mortality rate of 63.37%.The causes of death were multiple organ failure 45.50%and respiratory failure54.50%.Second partAfter mouse lung epithelial cell lines?MLE-12?are cultured in culture solutions with different PQ concentrations for 24h,the survival rate of MLE-12 is decreasing gradually along with the concentration of PQ.The concentration of PQ in IC50 is1015.72?mol/L,and that in all latter experiments is chosen to be 1000?mol/L.MLE-12 is treated for 24h in different groups.When the concentration of DXM is raised from 10.0?g/ml to 30.0?g/ml,the survival rate of the cells increases gradually.When it is raised to 30.0?g/ml,the survival rate reaches the highest,which is72%±2.87.When it is higher than 30.0?g/ml,that rate starts to decrease.For the purpose of studying the influence of DXM treatment on mouse lung epithelial cell induced by PQ,the following experiment groups are designed:NS group,PQ group,DXM group,PQ+DXM-L group,PQ+DXM-M group,PQ+DXM-H group.After MLE-12 cells are treated in groups as mentioned above,observation is conducted on the influence on cellular morphology first.When the PQ combination groups are added different dosages of DXM in,in PQ+DXM-L group and PQ+DXM-M group,the cells still have abnormal cellular morphology,all of which are suborbicular,the cellular state is poor,and the growth rate is slow,some of which are dying;when a high dosage of DXM is added in PQ+DXM-H,the cellular morphology is almost normal,and the growth is restricted to a certain degree.But compared with that in PQ,cells in PQ+DXM-L group and PQ+DXM-M group have a growth status closer to normal cells.Then,detection is done on the expression levels of SOD,MDA,and HYP in MLE-12 cell culture supernate.Compared with that in PQ group,SOD secreted by lung epithelial cell lines increases obviously while the expression levels of MDA and HYP decrease in PQ+DXM groups.Meanwhile,ELISA is adopted to detect the expression levels of TNF-?and IL-6.Compared with that in PQ group,the expression levels of TNF-?and IL-6 are also decreasing with the increase of DXM dosage.Later,the result of RT-qPCR shows that compared with that in PQ group,the expression levels of both TLR4 and NF-?B1 decrease to different degrees after they are induced by PQ and treated with DXM of different concentrations simultaneously.In same conditions,the expression levels of TNF-?and IL-6 decrease to different degrees compared with that in PQ group.The changes of protein level were detected by Western Blot.Compared with that in PQ group,the expression levels of TLR4was decrease after they are induced.The expression level of I?B-?is basically unchanged,and the change of p-I?B-?is obvious.Compared with NS group,PQ treatment led to a significant increase in p-I?B-?expression level,and the protein levels ofNF-?B1and p-NF-?B were also significantly increased.FCFM is used to detect the nuclear entry of NF-?B1 in PQ induced MLE-12 treated with DXM.Compared to NS group,the level of NF-?B1 in nucleuses after cells are treated with PQ increases and the level of NF-?B1 in nucleuses in PQ+DXM-H group decreases significantly compared with the PQ group.At last,NF-?B1 luciferase reporter system is used to prove that PQ induced MLE-12 cells after being treated with DXM can partially suppress the activation of NF-?B1 caused by PQ induction and the gene expression level regulated by NF-?B1 indeed.ConclusionOral dose of PQ,blood PQ concentration,urinary PQ concentration,blood SIPP,urine SIPP,white blood cell count?WBC?,Lac,blood PQ clearance time were different between survival group and death group.Blood purification treatment can improve the survival rate of patients.PICCO monitoring and lung function have a good effect on the the prognosis of PQ poisoning.Serum PQ concentration,24 hour EVLWI,blood SIPP and RV%TLC were independent predictors of PQ poisoning.DXM reduces PQ-induced mouse epithelial cell injury through the TLR4-NF-?signaling pathway.In the process of improving pneumocytes of the poisoned mice,the alleviating effect of DXM on lung injury would gradually attenuate after its concentration has exceeded a certain level.