Protective Effect And Mechanism Of Artesunate And Tetramethylpyrazine Combination On Experimental Cerebral Malaria Mice

Malaria is a major infectious disease that seriously threatens human health and life longevity.Cerebral malaria（CM）is the most serious complication caused by Plasmodium falciparum infection,and also one of the three major causes of death of children in Africa.In clinical practice,CM patients may have coma,disturbance of consciousness,meningeal irritation,hemiplegia,convulsions,ataxia and even death,and 10-17%of the surviving children have persistent neurological deficits.Among the pathogenesis of CM,the mechanical obstruction theory and the immunopathology theory are the two most widely recognized theories,which complement each other,but are still insufficient to explain the entire process of occurrence and development of CM[1,2].Pathological changes of CM include cerebral microvascular blockage,activation of immune cells,destruction of bloodbrain barrier,reduction of nitric oxide（NO）bioavailability,platelet activation,vascular inflaLmation,brain edema,etc[3-5].Previous research by our research team found that the combination of artesunate（AS）and tetramethylpyrazine（TMP）,an effective component of the blood activating and stasis removing drug Chuanxiong,can achieve better therapeutic effects on experimental cerebral malaria（ECM）models,and elevated NO levels play an important role in the drug efficiency.In addition to performing physiological functions through classical pathways,NO can also directly combine with proteins to play its role.The post-translational modification of proteins that can be modified through reversible oxidationreduction is called protein S-nitrosylation（SNO）,and the covalent bond bonds formed can be called S-nitrothiols（SNOs）[6-9]Previously,it was found that AS+TMP can simultaneously regulate multiple levels of S-nitroso protein,involving pathways such as energy metabolism,glycolysis,glutathione metabolism,and NMDA receptors.The key Snitroso protein is pyruvate dehydrogenase β（PDHB）was significantly elevated after AS+TMP treatment[10].Objective:In order to further verify the pharmacodynamic effect of AS+TMP during the critical phase of ECM and its impact on the corresponding NO and SNOs levels,this research will continue to use ECM model to study the therapeutic effect of AS+TMP during the critical phase of ECM,while clarifying the mechanism of its functional changes in Snitroso PDHB mediated by NO/redox system and NO.In view of the key role of NO in the intervention of AS+TMP in the acute episode of cerebral malaria,this study used NO eliminators to treat ECM mice and observed the changes in the therapeutic effect of AS+TMP,further verifying the mechanism of NO in the treatment of ECM by AS+TMP.The catabolism of S-nitrosoglutathione reductase（GSNOR）to nitrosoglutathione（GSNO）can inhibit NO mediated biological processes.In this study,GSNOR inhibitor N6022 was used to treat ECM mice to observe the changes in the pharmacodynamics of AS+TMP,further clarifying the impact of changes in S-nitrosoglutathione protein in vivo on the protection effects of AS+TMP.In order to further confirm whether AS+TMP can exert neuroprotective effects on ECM mice through S-nitroso modified PDHB mediated by NO/GSNO,this study constructed an adeno-associated virus（AAV）nitroso site mutant PDHB C263S to further elucidate the mechanism of AS+TMP’s efficacy through S-nitroso modified PDHB.To explore the synergistic effect of AS+TMP on ECM,this study used pharmacokinetics to analyze the changes in pharmacokinetic parameters of AS+TMP combination under physiological and pathological conditions,compared the advantages and disadvantages of intravenous and nasal administration,and used single drug groups AS,TMP,and AS+TMP to observe the synergistic effect of AS+TMP on pharmacokinetics.Method:In the first part,the ECM model was used to study the drug effect and its molecular mechanism.The experimental mice were randomly divided into five groups:Control group,Model group,tetramethylpyrazine group（TMP group,20.8 mg/kg）,artesunate group（AS group,15.6 mg/kg）,artesunate-tetramethylpyrazine group（AS+TMP group,20.8 mg/kg TMP and 15.6 mg/kg AS）.Statistics were made on the protozoaemia,body weight,body temperature,rapid coma and behavior scale（RMCBS）and survival curve of mice in each group.According to the level of protozoaemia in each group reaching about 10%,nasal administration was started（this time on the 5th day of inoculation）.HE staining and Periodic Acid Schiff Stain（PAS）staining were used to observe the pathological changes in the mouse brain,Fluoro Jade B（FJB）staining was used to detect neuronal degeneration in the mouse brain,and ELISA was used to detect the level of reactive oxygen species（ROS）in the mouse brain,The kit detects the ratio of nicotinamide adenine dinucleotide phosphate（NADPH）reduced to oxidized NADPH/NADP+and the ratio of glutathione（GSH）reduced to oxidized GSH/GSSG,and detects the expression of GSNOR and the activity of thioredoxin reductase（TrxR）,The activity of pyruvate dehydrogenase（PDH）and pyruvate kinase（PK）related to the key protein PDHB and the level of downstream acetyl coenzyme A（ACHA）were detected.The expression of phosphoglycerate dehydrogenase（PHGDH）,a key enzyme in serine synthesis,was detected.The parasitilized red blood cell（pRBC）human brain microvascular endothelial cell（HBMEC）model was constructed in vitro.IC₅₀ of AS+TMP was detected in vitro.The adhesion level of AS+TMP was determined by rosette assay,and the expression level of adhesion related molecule ICAM1 was detected.The activities of NO and NOS were detected by the kit,and the mRNA levels of Plasmodium enzymes related to S-nitrosoylation and glycolytic genes were detected.In the second part,after treating ECM mice with NO scavenger,observe the changes in the efficacy of AS+TMP,count the basic indicators of CM,HE,FJB,and PAS pathological staining in each group of mice,detect the relevant levels of NO and eNOS,detect the levels of redox related indicators ROS,NADPH/NADP+,and GSH/GSSG,detect the GSNOR expression and TrxR activity,the PDH and PK activities related to the key protein PDHB,and the level of downstream acetyl coenzyme A.The expression of PHGDH was detected by ELISA.In the third part,after treating ECM mice with GSNOR inhibitor N6022,the therapeutic efficacy of AS+TMP was tested.The basic pathological indicators,HE,FJB,and PAS pathological staining of ECM in each group were detected,and the levels of NO and eNOS were detected.The redox related indicators ROS,NADPH/NADP+,and GSH/GSSG were detected.The expression of GSNOR and TrxR activity were detected,and the activities of PDH and PK related to the key protein PDHB,as well as the downstream acetyl coenzyme A level,were detected,the expression of PHGDH was detected.The fourth part is to construct an adeno-associated virus（AAV）nitroso site mutant PDHB C263S.The vector GV388 was selected for enzyme digestion and linearization.The target gene PDHB C263S was amplified by PCR.The amplified product was connected to the vector and transformed.The positive transformants were identified by PCR reaction,and the positive clones were sequenced.Plasmid extraction and transfection were performed AAV PDHB C263S virus particles were collected for qPCR determination of viral titer.Next,the transfection mice experiment was conducted,and the mice were randomly divided into 7 groups,including 4 model groups:wild-type model group（WT+Model group）,negative control model group（AAV-scramble+Model）,adeno-associated virus mutation group（PDHB（C263S）group）,and adeno-associated virus mutation model group（PDHB（C263S）+Model group）;Three groups of drug addition groups:negative control drug addition group（AAV-scramble+AS+TMP）negative control model drug addition group（AAV-scramble+Model+AS+TMP）,adeno-associated virus mutation drug addition group virus model drug addition group（PDHB（C263S）+Model+AS+TMP）.After 4 weeks of intranasal instillation of the virus into mice,the mice were inoculated with Plasmodium berghei to construct an ECM model.According to the level of protozoaemia of about 10%,the administration of AS+TMP was started.The status of vector transfection and model construction was detected by immunofluorescence.The basic pathological indicators,HE and PAS pathological staining of the ECM in each group were detected,and the levels of redox related indicators ROS and eNOS were detected.The fifth part is the pharmacokinetic experiment of AS+TMP,constructing the LC-MS/MS method of AS+TMP,and conducting methodological validation.Firstly,the pharmacokinetics of physiological and pathological ECM mice was studied,and the logarithmic concentration time curve was obtained and the pharmacokinetic parameters were calculated;Next,an experiment was conducted to compare the pharmacokinetic differences between intravenous and nasal administration;Finally,compare the pharmacokinetic differences between the AS group,the TMP group,and the combined AS+TMP group.Results:In the first part,based on previous studies,this study used the ECM model to explore the pharmacodynamic effect,the REDOX level of ECM mice and the subsequent metabolic changes.AS+TMP can significantly reduce the death rate of mice,protect the liver and spleen and other organs,improve the malarial pigment deposition in the brain,and reduce the number of denaturetic neurons.When ECM occurs,the brain is in a state of peroxidation.The two redox regulatory factors NADPH/NADP+and GSH/GSSG decrease,the expression level of GSNOR decreases,the level of SNO-PDHB decreases,and the activity of PDH increases.The activity of PK decreases.Subsequently,the level of acetyl coenzyme A increases,and the expression of the key enzyme PHGDH in serine synthesis decreases.After administration of AS+TMP,ROS levels in vivo were significantly reduced,GSH/GSSG and NADPH/NADP+were increased,GSNOR expression level was decreased,SNO-PDHB expression was increased,PDH activity was decreased,acetyl coenzyme A content was decreased,and PHGDH expression was increased.AS+TMP can regulate redox regulatory factors in the body,improve related antioxidant metabolism levels,reduce excessive glycolysis caused by ROS,and play a role in protecting brain neurons and treating ECM.In vitro experiments,a model of parasitized red blood cell（pRBC）-human brain microvascular endothelial cell（HBMEC）was constructed to detect the antimalarial activity IC50 and cytotoxicity of AS+TMP in vitro,and the dose of AS+TMP administered in pRBCHBMEC was determined based on the results.Using the pRBC-HBMEC model,it was found that AS+TMP can reduce the expression levels of ICAM1,reduce the adhesion between Plasmodium parasites and endothelial cells,improve the levels of NO and NOS in HBMEC,and regulate the glucose metabolism of plasmodium and the expression of thioredoxin system genes at the transcriptional level.In the second part,the effect of AS+TMP on ECM mice was observed by treating ECM mice with NO scavenger.The results showed that the treatment of ECM mice with NO scavenger carboxy-PITO significantly weakened the protection of AS+TMP on liver and spleen and neurons,decreased the level of NO and eNOS expression in the mouse brain,and weakened the pharmacological effect of AS+TMP on neuroprotection.Using NO eliminators to observe the redox reaction in which NO participates,it was found that treating ECM mice with NO eliminators reduced the NADPH/NADP+and GSH/GSSG ratios compared to AS+TMP,while reducing the activity of TrxR,thereby affecting the redox reaction in vivo and weakening the antioxidant effect of AS+TMP.Further testing of subsequent metabolism,it was found that the drug could increase PDH activity,reduce PK activity and PHGDH expression,increase acetyl coenzyme A levels,increase glycolytic metabolism in vivo,and weaken the antioxidant metabolism of AS+TMP after treating ECM mice with NO scavenger.Comprehensive analysis showed that AS+TMP had a brain protective effect on ECM mice through the NO related redox system.The third part uses GSNOR inhibitors to indirectly increase the level of NO by increasing the content of GSNO,further verifying whether AS+TMP can exert brain protection and antioxidant metabolism through NO.The results show that GSNOR inhibitors enhance the insecticidal level and RMCBS score of AS+TMP after treating ECM mice;Significantly reduce the expression of TrxR and increase NADPH/NADP+,which can increase the level of NO and decrease the level of ROS as compared to using AS+TMP alone;When detecting PDHB related pathways,PK activity and PHGDH expression were significantly decreased,and there was no significant change in PDH activity;Compared to AS+TMP,the expression of acetyl coenzyme A was increased.The results showed that GSNOR inhibitor N6022 could significantly reduce glycolytic metabolism in the ECM brain of the drug group,and enhance the pharmacological effect of AS+TMP by increasing GSNO levels.In the fourth part,the PDHB peptide segments identified in S-nitroso proteomics were searched to confirm the corresponding S-nitroso sites.Then,based on the mouse PDHB protein transcript sequence in NCBI and the GPS-SNO1.0 nitroso site calculation tool,the S-nitroso modification site was jointly determined as Cys263.The PDHB nitroso point mutation vector was constructed,and sequencing showed that the vector was constructed correctly.The qRCR determined the titer of the vector,and the vector was used for animal model transfection.Immunofluorescence confirmed that the adeno-associated virus vector could be successfully expressed in the brain.By studying PDHB C263S mutant ECM mice,the effect of drug AS+TMP on the efficacy of ECM mice was investigated.The detection of ECM protozoaemia levels,body weight,body temperature,and RMCBS showed that the PDHB C263S mutation can affect the insecticidal level of AS+TMP,reduce the negative conversion rate of AS+TMP on ECM mice,and reduce the protective effect of AS+TMP on ECM mice;Through HE and PAS staining pathological analysis and brain tissue ROS detection,it was found that the PDHB C263S mutation can affect the protective effect of AS+TMP on ECM mice,and can aggravate the pathological symptoms of the hippocampus,liver,and lungs of ECM mice;Reversing the regulatory levels of ROS and eNOS in the brain tissue of ECM mice by drugs.The point mutation of PDHB C263S can inhibit the multiple pharmacological effects of AS+TMP.In the fifth part,a plasma and brain pharmacokinetic method for AS+TMP treatment of ECM mice was constructed using LC-MS/MS.In the methodological determination,the specificity,standard curve,lower limit of quantification,precision and accuracy,extraction recovery rate,and matrix effect were measured,and the results met the requirements.At the same time,a non compartment model was selected for parameter calculation.The comparison between ECM mice and normal mice showed that the AUC of AS and TMP in the brain of ECM mice was higher,and more drugs were injected into the brain.In the experiment of comparing the advantages and disadvantages of nasal administration and injection administration,the C_max of plasma AS and its active metabolite DHA is high during nasal administration,and AS and its active metabolite DHA can achieve relatively high blood drug concentrations,playing a highly effective role in killing insects;Intranasal administration of brain drugs AS and TMP has a higher AUC,which can target the brain of ECM mice and better exert the pharmacological effects of AS.In the synergistic effect experiment,the AS+TMP group significantly increased the AUC of AS,DHA,and TMP in the brain,and increased the C_max of DHA and TMP in the brain.The combination of AS+TMP can significantly increase the amount of AS and TMP in the brain.AS+TMP has a synergistic pharmacokinetic effect in the treatment of ECM.Conclusion:This study elucidates the pharmacological effects and mechanisms of drug AS+TMP on ECM during the critical phase.The combination of AS and TMP can effectively improve the pathological damage,affecting the NO related redox system in ECM mice.Using NO scavenger,GSNOR inhibitor,PDHB S-nitrosation point C263S point mutation,the results showed that AS+TMP could affect the balance of NO related redox system through PDHB S-nitrosation,and play a pharmacodynamic role in improving the pathological damage of ECM during the critical phase.By comparing the plasma and brain pharmacokinetic parameters of nasal administration and intravenous injection,it was confirmed that nasal administration can better exert brain targeted and rapid insecticidal effects.By comparing the plasma and brain pharmacokinetic parameters between the single drug group and the AS+TMP combination group,the synergistic effect of AS+TMP combination on pharmacokinetics was verified.