| ObjectiveAcute pesticide poisoning has become a public health and social problem of global concern,and its clinical diagnosis and precise treatment has always been the research field of medical researchers.At present,acute pesticide poisoning is mainly caused by organophosphorus and paraquat.The toxicity of paraquat is severe,there is no specific antidote,the poisoning harm is great,and the mortality is very high.Although paraquat was banned,there are still illegal sales and use,and acute poisoning incidents caused by misuse also occur from time to time.Therefore,the purpose of this study is to establish a quantitative detection method for common acute pesticide poisoning,including organophosphorus and paraquat,to study the toxicological mechanism of paraquat poisoning by metabonomics,to find the potential biomarkers of paraquat poisoning,reveal its toxicological mechanism from the metabolic level and predict the therapeutic targets,to study the molecular biological mechanism of paraquat and the therapeutic effects of paeonol and its mechanism in vivo and in vitro.All these studies can provide strong technical support for the clear diagnosis of acute pesticide poisoning,and lay a foundation for the further study of the toxic mechanism of paraquat and the development of effective drugs for the treatment of paraquat poisoning.Methods1.Establishment of quantitative detection method and technical platform for plasma toxicants in patients with acute pesticide poisoning.The detection pesticides and methods were screened according to the poisoning frequency and the physicochemical properties of the pesticide.(1)Paraquat and diquat were simultaneously determined by HPLC-DAD.The plasma were treated using Waters OASIS(?)column,then separated on a Thermo Hypersil Gold(250×4.6 mm,5 μm)column with the mobile phase consisted of 75 mmol/L sodium heptane sulfonate(containing 0.1 mol/L phosphoric acid,pH3.0)and acetonitrile(87:13,v:v)at a flow rate of 1.0 mL/min.The full-wavelength scanning was 200-400 nm,the detection wavelength of paraquat and diquat were 257nm and 310nm,respectively.The plasma samples obtained from the suspected acute pesticide poisoning patients were collected and analyzed by the established method.(2)The pesticides(including dichlorvos,acetochlor,atrazine,chlorpyrifos,a-endosulfan,and β-endosulfan)were determined by GC-MS.The plasma was treated with acetonitrile,and concentrated using anhydrous sodium sulfate.Borneol was used as internal standard.The separation was performed on a HP-5MS capillary column(30 m×0.25 mm×0.25μm)with temperature programming.The detection was performed under electro-spray ionization(ESI)in selected ion monitoring(SIM)mode.The plasma samples obtained from the suspected acute pesticide poisoning patients were collected and analyzed by the established method.Continue to expand the types of poison detection,establish qualitative and quantitative detection technology platform for clinical and patients.2.The metabonomics of paraquat poisoning in human plasma were studied by UPLC-MS,to explore the toxicological mechanism,potential markers and therapeutic targets of paraquat poisoning.The plasma sample was precipitated with methanol,then separated on Thermo Hypersil GOLD(100 x 2.1 mm,1.9 um)column with the mobile phase consisted of 0.1%formic acid aqueous solution and 0.1%acetonitrile formate at a flow rate of 0.3 mL/min.The obtained data were analyzed by PCA and PLS-DA,which were multivariate statistical analysis methods.Differential metabolites were screened and analysed using the online analysis platform of MetaboAnalyst 4.0.3.The therapeutic effect and mechanism of paeonol on paraquat induced pulmonary toxicity were studied in vitro and in vivo.(1)Cell experiment:Human lung fibroblast(HFL1)cells were used in this study.CCK8 method was used to select the best concentration of paraquat and paeonol,and investigate the effect of paraquat and/or paeonol on cell proliferation.The cell apoptosis was detected by V-FITC/PI double staining,reactive oxygen species(ROS)was detected by DCFH-DA probe,and the mRNA expressions of SOD,TNF-α and IL-1β were detected by RT-PCR.The protein expressions of Nrf2,NF-κB,Bcl 2,Bax and cleaved-Caspase 3 were detected by Western-blot.(2)Animal experiment:45 Balb/c mice were randomly divided into control group,paraquat group(100mg/kg)and paraquat(100mg/kg)+paeonol group(50mg/kg).After paraquat was intragastrically administered,paeonol was also intragastrically administered at 2h,24h,48h and 72h.The mice were further observed for ten days and killed on the eleventh day.The lungs were taken,weighed,and the lung W/D value was calculated.GSH,MDA,SOD,TNF-α and IL-1β in lung tissue were detected by ELISA method.The histopathological changes of lung tissue were observed by HE staining.The apoptosis of lung was evaluated by Tunel method.The protein expressions of Nrf2,NF-κB,Bcl 2,Bax and cleaved-Caspase 3 were detected by Western blot.Results1.Establishment,validation and clinical application of quantitative detection method for plasma toxicants in acute pesticide poisoning patients.(1)The calibration curves of HPLC-DAD method for paraquat and diquat ranged from 0.05 to 20 μg/mL,and the precision of LLOQ for paraquat was 16.49%which was required to be less than 20%.The precision of other concentrations was less than 14.14%.The recovery of paraquat and diquat was 95.38-103.97%and 94.79-98.40%,respectively.The results showed that paraquat and diquat were stable under various storage conditions.In the plasma samples of suspected acute pesticide poisoning patients,paraquat was detected in 127 samples with the concentration range of 0.10-20.62 μg/ml(mean 3.60μg/ml),diquat was detected in 24 samples with the concentration range of 0-26.59μg/ml(mean 4.72 μg/ml).Paraquat and diquat were simultaneously detected in 5 samples.(2)Good linear ranges of 0.05-10 μg/mL were obtained for all the analyzed pesticides determined by GC-MS method.The linear correlation coefficients were greater than 0.99.The average recoveries were determined between 86.8 and 106.5%.The inter-and intra-day precisions were in the ranges of 1.7-14.5%and 4.2-13.8%,Dichlorvos was unstable in plasma both at room temperature and freezing conditions.It can be degraded by 50%after 2 hours at room temperature,20%after 2 freeze-thaw cycles,15%to 20%after 7 days and 17 days at-20℃.The rest of storage conditions are stable.Other five pesticides were stable after storage at-20℃ for 17 days and two freeze-thaw cycles.Among the plasma samples of suspected acute pesticide poisoning patients,dichlorvos were detected in 13 samples with mean concentration of 0.289 μg/ml,atrazine were detected in 6 samples with mean concentration of 0.261μg/ml,acetochlor was detected in 1 sample with concentration of 0.153 μg/ml.Endosulfan and chlorpyrifos were not detected.Paraquat was the most frequently detected in clinical samples,followed by diquat and dichlorvos.2.Fourteen differentially expressed metabolites,including 5-Chloro-4-oxo-L-norvaline,Phenylalanine,3,4-dihydroxyphenylacetic acid,linoleyl carnitine,Lyso PE(0:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)),PC(18:3(9Z,12Z,15Z)/18:2(9Z,12Z)),arachidonic acid,D-Sphingosine and bilirubin,were found by metabonomics studies.Metabolic pathway analysis showed that paraquat poisoning could affect the biosynthesis pathway of phenylalanine,tryptophan and tyrosine,and affect the metabolic pathway of arachidonic acid,phenylalanine,glycerophospholipids,porphyrin and chlorophyll metabolism.It is suggested that paraquat poisoning may interfere with the oxidative stress and energy metabolism of human body,and cause the injury of human lung,liver and kidney.3.Paeonol can alleviate the damage of HFL1 cells and mice lung by inhibiting Nrf2 and activating NF-κB protein expression.(1)Cell experiment:The results of CCK8 method showed that the optimal concentration of paraquat and paeonol on HFL1 cells were 150μm and 50μm,respectively.Paraquat could significantly inhibit the proliferation of HFL1 cells,while paeonol could weaken the inhibition of paraquat.After 24 hours of treatment with paraquat and/or paeonol,the apoptosis rates of paraquat group and paraquat+paeonol group were 22.96%and 11.76%,respectively,and the difference was statistically significant.The level of ROS in paraquat+paeonol group was significantly lower than that in paraquat group.RT-PCR results showed that compared with the control group,the expression level of SOD mRNA in paraquat group was significantly lower,and the expression levels of TNF-α and IL-1β mRNA were significantly higher,while the expression level of SOD mRNA in paraquat+paeonol group was significantly higher,and the expression levels of TNF-αand IL-1β mRNA were significantly lower than that in paraquat group.Compared with the control group,paraquat increased the protein expression of NF-κB(p65 and p-p65),Bax,cleaved-caspase 3,and decreased the expression of Nrf2 and Bcl 2.Compared with paraquat group,in paraquat+paeonol group,the activity of p65,p-p65,Bax,cleaved-caspase 3 protein was inhibited,and the activity of Nrf2 and Bcl 2 protein was enhanced.(2)Animal experiment,compared with the control group,the lung W/D value and pathological score of lung injury in paraquat group were significantly increased,and the lung W/D value in paraquat+paeonol group was significantly lower than that in paraquat group.HE staining results showed that the lung tissue structure in paraquat group was significantly damaged,with severe thickening of alveolar wall and significant hyperplasia of connective tissue,accompanied by a large number of lymphocytes,neutrophils and oligodendrocytes infiltration.Compared with the control group,the apoptosis of paraquat group was significantly increased,however,the apoptosis of paraquat+paeonol group was significantly improved.Compared with the control group,GSH and SOD in paraquat group were significantly decreased,and MDA was significantly increased.Compared with paraquat group,GSH and SOD in paraquat+paeonol group were significantly increased,and MDA was significantly decreased,the difference was statistically significant(P<0.05).Compared with the control group,the concentration of TNF-αand IL-1β in paraquat group was significantly increased,but in paraquat+paeonol group,the concentration of TNF-α and IL-1β was significantly decreased compared with paraquat group.The results of western blot showed that,compared with the control group,the protein expression of p65,p-p65,Bax,cleaved-caspase 3 in paraquat group was significantly increased,and the expression of Nrf2 and Bcl 2 was significantly decreased.Compared with paraquat group,the expression of p65,p-p65,Bax and Cleaved-caspase 3 in paraquat+paeonol group was significantly decreased,and the expression of Nrf2 and Bcl 2 was significantly increased.Conclusions1.The methods of HPLC-DAD and GC-MS established in this study have the advantages of high throughput,high sensitivity,simple operation and wide linear range which can be used for the screening and concentration determination of suspected paraquat,diquat,dichlorvos,acetochlor,atrazine,chlorpyrifos,endosulfan and other poisons.It can provide a clear basis for clinical diagnosis of acute pesticide poisoning,and provide important basis for diagnosis,precise treatment and prognosis evaluation.It is helpful to further increase and expand the types of poison detection,enrich and improve the qualitative and quantitative detection technology platform for clinical and patients.2.The potential biomarkers and the metabolic pathways that may be affected by paraquat poisoning were found by the metabonomics study of acute paraquat poisoning.It can be used to assist the diagnosis of paraquat poisoning and provide therapeutic targets,suggesting that clinical treatment measures can be taken to restore the biosynthesis or metabolic level of arachidonic acid and amino acids as soon as possible and improve the success rate of clinical treatment.3.In vivo and in vitro studies have confirmed that paraquat can induce inflammation and apoptosis in HFL1 cells and mice lung,which may be related to inhibiting Nrf2 and activating NF-κB signaling pathway.Paeonol can alleviate the inflammatory reaction and apoptosis of HFL1 cells and mouse lung induced by paraquat through activating Nrf2 and inhibiting NF-κB signaling pathway,and alleviate the damage of HFL1 cells and mouse lung caused by paraquat.It suggests that paeonol has potential value in clinical treatment of paraquat poisoning,which can be further verified by clinical studies. |
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