Exploratory Study Of Artesunate And Tetramethylpyrazine Combined Therapy For Cerebral Malaria Based On Improvement Of Neurological Damage In Experimental Cerebral Malaria

Background and objective Cerebral malaria(CM)is the most deadly neurological complication caused by Plasmodium falciparum infection.CM occurs mostly in children under 5 years of age in Africa,accounting for 90% of total cerebral malaria deaths,even if surviving through antimalarial treatment,10% to 20% of children still have neurological sequelae,causing severe neurological deficits,including learning and memory disorders,language barriers,and behavioral disorders.At present,intravenous antimalarial artesunate(Art)is still the main drug for the treatment of CM,but the use of Art alone can not improve the neurological sequelae.Furthermore,the patients in poor African countries usually do not timely access within 24 hours to Art,leading to number of deaths.In this study,Tetramethylpyrazine(TMP),an extract from Ligusticum chuanxiong,a blood-activating and stasis-removing drug,was combined with Art as a new compound to explore the therapeutic effect of neuroprotection on CM and iTRAQ analysis was performed to provide the basis for further studies on mechanism of action of drugs,and to assist in identifying potential biomarkers for monitoring disease improvement of Plasmodium infection.Research methods The experimental cerebral malaria(ECM)model was established by infecting C57BL/6mice with Pb ANKA(Plasmodium berghei ANKA),and the experimental mice were divided into model group,Art treated group,TMP treated group,Art+TMP combination group.The survival rate,body weight and temperature of mice in each group were counted by intranasal administration on the 2nd to 4th day after inoculation,and parasitemia was counted by Giemsa staining;Rapid murine coma and behavior scale(RMCBS)was used to evaluate the course of disease in ECM mice and the behavioral performance of ECM mice was further evaluated by the classical behavioralexperiments of mice – open field and Y maze test.The cerebral vascular occlusion was observed by hematoxylin and eosin(HE)staining.The cerebral vascular blood flow changes and vascular integrity were observed by MRI.Immunohistochemical method was used to evaluate the expression levels of adhesion moleculesvascular cell adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)and glial fibrillary acidic protein(GFAP)and neuron-specific nuclear protein(NeuN).The expression levels of brain-derived neurotrophic factor(BDNF),neurotrophic factor-3(NT-3)and nerve growth factor(b-NGF)and vascular endothelial growth factor A(VEGF-A),inflammatory factors TNF-α and IL-10 were detected by mouse cytokine chip and ELISA.Toxicological study of intranasal administration was detected by HE staining.The protective mechanism of Art+TMP combination in ECM mice was further analyzed by iTRAQ’s quantitative proteomics.The potential of neuroprotective effects of Art+TMP in ECM mice was analyzed by gene function enrichment analysis and protein interaction analysis.The mechanism of action and further verification of the accuracy of the data by immunoblotting.Research results The combination of Art and TMP can improve the survival rate of mice,prevent the occurrence of neurological symptoms,significantly improve the body weight,body temperature drop caused by CM,clinical symptoms such as ataxia,hemiplegia and coma.The open field and Y maze experiments show Art and TMP combination can significantly improve the motor ability and exploration ability of ECM mice.These effects are associated with decreased expression of adhesion molecules ICAM-1,VCAM-1 in the brain,decreased expression of GFAP,increased expression of neuronal nucleoprotein NeuN,increased expression of neurotrophin and decreased expression of pro-inflammatory cytokines,and maintenance of vascular integrity.and reducing the infiltration of inflammatory cells in the brain of ECM mice is associated withobstruction of pRBC.At the same time,the toxicological results of intranasal administration showed that intranasal administration was safe and feasible.These results suggest that Art and TMP combination may be a source of neuroprotection and immune regulation of ECM and may be used as an antimalarial drug to improve the treatment of CM.To elucidate the possible targets for the combined treatment of ECM with Art and TMP combination,According to the relative quantitative proteomics(iTRAQ),the expression folds were changed by 1.2 times or more,and the p value was <0.05,and217 down-regulated proteins and 177 up-regulated proteins were identified.The proteome characteristics of the Art and TMP combination groups were significantly altered compared to the Art or TMP alone treatment group.The neuroprotective effects of Art and TMP in combination with functional enrichment analysis and protein interaction may be related to axonal development or blood-brain transport processes.Conclusions The combination of Art and TMP can effectively treat the clinical symptoms of ECM and exert neuroprotective effects,which is expected to be an adjuvant therapy for clinical CM.iTRAQ proteomics provides a resource for further study of the synergy between Art and TMP and provides a potential prognostic biomarker for this therapeutic intervention.