| Objective:Organophosphorus compounds were developed in the early 20th century and mainly used as pesticides to protect crops[1].Acute organophosphate poisoning is one of the common poisonings in emergency department.The main poisoning routes include oral and respiratory tract inhalation and ingestion,and skin contact[2].Most of the organophosphate poisoning patients come from occupational exposure and suicide[3][4].Organophosphate poisoning has a wide range of toxic effects on the liver,lungs,kidneys,brain and heart[8].The main mechanism of organophosphate poisoning is by inhibiting the activity of acetylcholinesterase,leading to a large accumulation of acetylcholine in the central and peripheral nervous system.Excessive acetylcholine can cause severe toxicity,stimulate muscarinic and nicotinic receptors,and cause bile Alkaline syndrome（within 24h）,diarrhea,urinary incontinence,miosis,bradycardia,bronchospasm,salivation,tearing,vomiting,hypotension,arrhythmia and other muscarinic symptoms,fasciculations,muscle weakness,Hypertension,tachycardia,sweating,enlarged pupils and other nicotinic symptoms[5][6].Later,during the strict treatment period,Intermediate Syndrome（IMS）may occur（24h-7d）,paralysis of respiratory muscles,extreme muscles,neck flexors,motor cranial nerves and neuromuscular junction（NMJ）dysfunction,and due to nerve targets Delayed neuropathy（OPIDN）occurs due to the inhibition of esterase（NET）and aging（7-21d）[7].In the early stage of organophosphate poisoning,in the past sixty years,the clinical care of patients with organophosphate poisoning has hardly improved.The current clinical medicine used to treat organophosphate poisoning is still atropine+oxime[9].But the shortcomings of these two treatments are also obvious.Excessive use of atropine can lead to tachycardia,high fever,delirium and intestinal dysfunction,paralytic intestinal obstruction[10].There is no clear ideal dosage regimen of atropine,and the detoxification effect of oximes in the treatment of organophosphate poisoning also exists.Discussion and questioning,oximes can not protect and reactivate the acetylcholinesterase of the central nervous system[11].At present,this treatment method is considered unsatisfactory and inadequate.Under such circumstances,there is an urgent need for a more efficient,new type of drug with less side effects to treat organophosphate poisoning.In 1953,Mazur[12][13]found this enzyme capable of hydrolyzing paraoxon in the tissues of mammals（but not found in birds,reptiles,and fish）.Enzyme),so it was named paraoxonase.Because of its high-efficiency and non-toxic hydrolysis of various organophosphorus compounds in vivo and in vitro,it has attracted widespread attention in related research.PON1 is a calcium-dependent enzyme,composed of 354 amino acids,with a molecular weight of about 43KDa[14].PON1 is mainly produced in the liver of the human body and released into the blood.Because it has a hydrophobic signal sequence in the N-terminal region,it can bind to HDL[15]and perform multiple functions,including preventing the oxidation of low-density lipoproteins,thereby reducing arteries The development of atherosclerotic plaques and the detoxification of various OP compounds[16].Immunohistochemical studies have found that PON1 exists in many tissues in mammals,but it is not known whether it is synthesized by the tissue itself or transported to the tissue through HDL[17].The human PON1 gene is located on the long arm of chromosome 7（7 q 21.3-22.1）and has a six-blade propeller structure.The coding sequence includes 9 exons and 8introns,while the mouse PON1 gene is located on the 6th The proximal region of the chromosome[18].In different humans,the activity of PON1 can vary by 10-40 times,which is determined by the genetic polymorphism of PON1.The PON1-Q192R polymorphism in the coding region affects the catalytic activity of PON1,while the PON1-L55M polymorphism in the coding region and the PON1-T108C polymorphism in the promoter region affect the expression level of PON1 in serum[19].In addition,individuals with different PON1 genotypes have different susceptibility to different organophosphorus compounds.Among them,the susceptibility of PON 1 to dichlorvos,chlorpyrifos,and paraoxon is lower than that of type Q,while the susceptibility of type Q to diazinon and sarin in PON 1 is lower than that of type R.In other words,the two gene subtypes of Q//R at position 192 of PON 1 have different hydrolysis effects on different types of organophosphorus compounds[20].In previous experiments,we used genetic engineering technology to prepare recombinant human PON1Q192 subtype using E.coli as an expression system[21].However,the yield and activity of recombinant PON1 produced by this prokaryotic expression system are relatively low,and during the production process,many expressed enzymes will eventually aggregate to form inclusion bodies,and the protease in the fermentation broth needs to be renatured to ensure the yield[22,23].In the process of renaturation,a large amount of enzyme protein will be lost,and the activity of the enzyme will be greatly reduced.Second,the use of prokaryotic cells to express eukaryotic proteins lacks the post-translational modification process that eukaryotic proteins should have[24].Therefore,the protein expressed by the target gene lacks necessary glycosylation,sulfhydrylation,phosphorylation and other modifications,which in turn affects the tertiary spatial structure of the protein,and affects or even changes the activity of the protein[25].Through previous research,we found that large-scale suspension culture of baculovirus-insect cells is a very efficient eukaryotic protein expression system.At the same time,it meets the requirements of post-translational glycosylation and phosphorylation,making the expressed protein closer to the natural protein in structure and function.With the aid of baculovirus carrying PON1 gene subtype to transfect insect cells-SF21[27],the target gene can be expressed on a large scale in a bioreactor.Its protein expression efficiency is high,the yield is large,and the enzyme activity is stable,which is a very ideal expression system[28].However,in the actual preparation process,although the yield and activity of recombinant PON1 have been improved compared with previous reports,it still cannot meet the needs of subsequent experiments.Thirdly,the prepared PON1 with different gene locus polymorphisms lacks targeted substrate dependence research[26],so the detoxification effect of different genotypes of PON1 on different substrates is not clear.The insect cell-baculovirus expression system needs to be improved to obtain more and more active recombinant PON1.The Baculovirus Expression Vector System（BEVS）is a transient expression platform for the production of recombinant proteins in insect cells.Baculovirus infection of insect cells will cut off host translation and induce apoptosis,and cause the termination of protein expression.This limits the expression of the target protein to 1-3 days after transfection.Seriously affect the yield of the target protein.Therefore,this experimental study intends to use RNA interference technology to construct a recombinant baculovirus pIBds Casp-1 containing Sf-caspase-1 inverted repeat sequence,and then transfect SF21 cells to transform the host cells to cultivate RNAi-mediated Sf-Stable cells inhibited by caspase-1.Inhibit the expression of SF-Caspase-1 in host cells,delay cell death and lysis,and improve the output and activity of recombinant protein expression.With sufficient recombinant PON1,relevant research on dephosphorylation can be carried out to find the effect and mechanism of post-translational modification on PON1 activity[29].Finally,the detoxification mechanism of PON1 Q/R isoenzyme pairs of two genotypes of PON1 Q/R was further discussed in detail through animal experiments.Method:1.Construction of recombinant baculovirus pIBds Casp vector.Use the pIB vector as the backbone to construct a stable transfection plasmid.The 5’end 800 base pair（bp）forward and reverse DNA fragments of Sf-caspase-1 gene were amplified from genomic DNA extracted from Sf21 cells by polymerase chain reaction（PCR）.The resulting construct is named pIBds Casp and contains the inverted repeat of Sf-casp1under the control of the opIE2 promoter.After more than 20 passages,the stable cell line was analyzed by genomic DNA PCR and RT-PCR to check the inverted repeat DNA sequence of Sf-caspase-1 and the amount of endogenously expressed Sf-caspase-1m RNA.Confirmed to form a stable SF21 cell line.Then,the recombinant baculovirus containing the target gene PON1 was used to transfect the modified stable SF21 cell line.After 5 days of culture,the protein was recovered and purified by nickel column chromatography.The activity of the target protein is detected and compared by spectrophotometry.2.To study the effect of dephosphorylation on the activity of rhPON1192R/Q isoenzyme and the hydrolysis of organophosphorus compounds.Use alkaline phosphatase to dephosphorylate rhPON1 R192,rhPON1 Q192 and human mixed serum（h PON1 mix）.Select the three most commonly used iconic organophosphorus pesticides in China:dichlorvos（high toxicity）,dimethoate（toxic）,and trichlorfon（low toxicity）as the substrates and divide them into three groups.Before and after dephosphorylation,rhPON1 192R/The enzyme activity changes of Q and h PON1 mix and the changes in the in vitro hydrolysis activity of the above three organophosphorus substrates,and the differences in the hydrolysis activities of the three organophosphorus substrates were compared.Using electrophoresis and Western Blot detection methods,compare the possible changes in the molecular weight and molecular structure of rhPON1 192R/Q and h PON1mix before and after dephosphorylation,and explore the importance of post-translational modification for maintaining the activity and substrate specificity of human PON1 192R/Q effect.3.To study the protective effect of the two isoenzymes of rhPON1 192R/Q isoenzymes on the lung tissue of rats with organophosphate poisoning144 SD rats were randomly divided into treatment group A（rhPON1 R192 treatment group）,group B treatment（rhPON1 Q 192 treatment group）,group C treatment group,and group D rhPON1 intervention control group.According to the type of organophosphorus poison（dichlorvos,dimethoate,trichlorfon）,each group was divided into four subgroups,each with 9 rats.The animal poisoning model was established by gavage.In each subgroup,the poison was exposed to 1.5 times the lethal dose（1.5×LD50）of the poison.Among them,for group A and group B,intraperitoneal injections of rhPON1 192R and rhPON1 192Q were given at a dose of 10 U/kg within 1 minute after exposure to detoxification.Continuously observe and record the time and degree of the symptoms of poisoning in each group of rats,including salivation time,systemic muscle tremor time,apparent dyspnea time,muscle strength score and other indicators.After 12hours of continuous observation,it was set as the end of the experiment.All rats were sacrificed by the method of cervical dislocation,and serum and brain tissue samples were collected immediately.Spectrophotometric method was used to measure and compare the changes of cholinesterase activity in serum and lung tissue of the poisoned group and the treatment group,and the methods of light microscope and transmission electron microscope were used to observe and compare the ultramicroscopic lung tissue cells and cell nucleus in the poisoned group and the treatment group Structural changes,Western Blot and immunohistochemical methods to detect and compare the expression of cholinesterase in lung tissues in the infected group and the treatment group,and compare the specificity of different PON1 gene isoenzymes to the substrates of different organophosphate poisons To study the factors that lead to the specificity of this substrate,and to find isoenzymes of PON1 gene subtypes that efficiently hydrolyze different organophosphorus poisons.4.To study the difference in the hydrolysis activity of R/Q isozymes of rhPON1 on organophosphorus compounds containing P-O bonds and P-S bonds.We found in previous experiments that the R/Q isoenzymes of paraoxonase 1 have poor detoxification effects on trithion poisoning.It may be because PON1 has a weaker hydrolysis effect on the PS bond than PO For some organophosphorus poisoning containing PS bonds,paraoxonase may not be a good detoxification option.The reason is probably related to the firmness of the phosphorus-sulfur bond.We have carried out further research on the comparison of the hydrolysis effect of the phosphorus-sulfur bond and the phosphorus-oxygen bond.We have selected several pesticides commonly used in agricultural production in my country,and divided them into P-O bond group and P-S bond group according to their chemical structure.Then compare the hydrolysis effects of the R/Q isozymes of paraoxonase 1 on the two types of organophosphorus compounds.Result:1.Through PCR and RT-PCR on the modified SF21 cells,the results show that the pIBds Casp vector has been stably integrated with the genomic DNA of Sf21 cells.In addition,the data also showed that the level of Sf-Casp1 m RNA in Sf21/pi Bds Casp-1cells was significantly lower than that observed in Sf21 and Sf21/pi B cells.Sf-caspase-1ds RNA successfully inhibited the expression of Sf-caspase-1 m RNA in Sf21/pi Bds Casp-1 cells.2.Use rhPON1 192Q-P2-Vir and rhPON1 192R-P2-Vir virus to infect sf21,Sf 21/pi B and Sf 21/pi Bds Casp-1 cells at a multiplicity of infection（MOI）of0.1,1,and 10,respectively.At 2-4 dpi,the cumulative expression of recombinant PON1in r Bac SEAP-infected Sf21/pi Bds Casp-1 cells was significantly higher than that in the control group in all MOIs.When the MOI was 1 and 10,the cumulative expression of recombinant PON1 in Sf21/pi Bds Casp-1 cells infected with virus after 4 dpi was about 2times higher than that in the control group.The difference in cumulative recombinant PON1 expression between normal and Sf-caspase-1 inhibited stable cells increased with the increase of MOI.These data indicate that after baculovirus infection,the production of recombinant protein PON1 secreted by stable cells inhibited by Sf-caspase-1 is higher than that of normal SF21 cells.When MOI=10,the expression time of recombinant PON1 is early,the cumulative expression is high,and the expression is high at 24h.3.After conducting hydrolysis experiments on different organophosphorus substrates,it was found that in the hydrolysis activity of dichlorvos,rhPON1 R192>h PON1 mix>rhPON1 Q192,and the hydrolysis activity of trichlorfon showed rhPON1 R192<h PON1mix<rhPON1 Q192 For the hydrolysis activity of dimethoate,rhPON1 R192>h PON1mix>rhPON1 Q192,but the hydrolysis activity was significantly weaker than that of dichlorvos and trichlorfon groups.After dephosphorylation,the hydrolysis activity of each group showed a significant decrease,but showed the same substrate specificity as before dephosphorylation,showing that phosphorylation plays an important role in maintaining the activity of recombinant proteins.4.By comparing the chemical properties and molecular structures of dichlorvos,trichlorfon,dimethoate,and finding that their molecular structures are somewhat different.The molecular structures of dichlorvos and trichlorfon contain P-O bonds,while the molecular structures of trithion and dimethoate contain P-S bonds.The P-S bond is stronger than the P-O bond.The chemical properties of trithion and dimethoate are relatively stable,which is related to their P-S bond.5.Animal experiments compared the detoxification effects of recombinant PON1 against dichlorvos,trichlorfon and trithion in vivo.The results showed that the hydrolysis effects of the two isoenzymes of PON1 gene subtypes on different types of organophosphorus compounds may be completely opposite.The hydrolysis effect of PON1 R192 on dichlorvos is better than that of PON1 Q192,while the hydrolysis effect of PON1 R192 on trichlorfon is weaker than that of PON1 Q192.6.From the results of HE staining of lung tissue,it can be seen that dichlorvos,trichlorphon,and dimethoate are poisoned.Obvious pulmonary edema and alveolar septum swelling in the rhPON1 192R/Q treatment group were accompanied by a large number of inflammatory cell infiltrations.In the rhPON1 192R/Q treatment group,pulmonary edema and alveolar septum swelling were significantly reduced,and inflammatory cells were reduced.The AchE activity results of serum and lung tissue homogenates showed that compared with the control group,the AchE activity of the poisoned group was significantly decreased,while the AchE activity of the rhPON1 192R/Q treatment group was significantly higher than that of the poisoned group.The results of the levels of SOD,GSH-Px and MDA in lung tissues showed that compared with the control group,the SOD and GSH-Px of the poisoned group decreased significantly,and the MDA increased,while the SOD level of the rhPON1 192R/Q treatment group was higher than that of the poisoned group.At the same time,the level of MDA decreased.These results indicate that rhPON1 192R/Q can not only protect lung injury caused by organophosphate poisoning by increasing the level of AchE activity in lung tissue through the well-known cholinergic mechanism,but also can act as an antioxidant through non-cholinergic mechanisms.It reduces the oxidative stress damage caused by organophosphorus poisoning and protects the lung tissue.Conclusion:1.Using RNA interference technology,stable SF21 cells inhibited by Sf-caspase-1 can be obtained.At 2 and 3 days after infection,the inhibition of apoptosis enhanced the production of recombinant PON1 protein.Five days after infection,compared with cell lines not modified with si RNA,the activity of the protein in stable SF21 cells inhibited by Sf-caspase-1 is still higher,and the target protein production is also higher.2.Phosphorylation plays an important role in maintaining the activity of paraoxonase 1.Before dephosphorylation,rhPON1 192R/Q and natural h PON1 showed the same enzymatic activity and specificity for substrate hydrolysis.After dephosphorylation,the molecular weights of rhPON1 192R/Q and the natural h PON1showed a consistent decrease,and the enzyme activity also showed a significant decrease.This decrease in activity will not be different due to changes in the substrate.,Rh PON1192R/Q and natural h PON1 have no specificity for substrate hydrolysis.3.rhPON1192R/Q has a certain protective effect on the lung tissue of rats exposed to different organophosphorus pesticides,and protects the respiratory system by reducing the inhibition of organophosphorus poisons on the cholinesterase in serum and lung tissue.For different organophosphorus poisonings,the therapeutic effects of rhPON1 192R/Q are significantly different.4.PON1 has better hydrolysis effect on P-O bond than P-S bond.This may be because the P-S bond may be stronger than the P-O bond,making the organophosphorus compound containing the P-S bond less likely to be hydrolyzed by PON1. |
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