Therapeutic Effect And Mechanism Of Artesunate Combined With Xingnaojing On Monkey Cerebral Malaria

ObjectiveMore than 3 billion people are infected with malaria in 109 of the world’s malaria endemic countries,especially children under five years of age and non-immunized adults.1%-2%of patients infected with falciparum malaria will develop severe malaria.The most serious complication is Cerebral Malaria(CM),which is an important cause of death and neurological diseases.Cerebral malaria is a coma that can detect asymptomatic malaria parasites-that is,lethargic,only painless,no locating,or deeper coma.Cerebral malaria is due to the parasite blockade of Plasmodium falciparum,adhesion,retention in the microvascular vein side,resulting in vital organs of ischemia,hypoxia and damage.If this time with artemisinin-like drug treatment,of course,the protozoa can be quickly killed,however,after the death of the big nourishing body,schizophrenia is still blocked in the microvascular,continued or even prolonged damage to vital organs,often resulting in dead.Therefore,to reduce the mortality of cerebral malaria,the key is to actively look for a new method,as soon as possible to lift the erythrocyte adhesion,and lift microvascular tissue damage because of blocking.The existing western medicine treatment method can not solve the protozoa adhesion obstruction caused by cerebral malaria death,which makes the disease mortality is high.Cerebral malaria is similar with Zhangnue in Chinese medicine,which is due to miasma infection and Wen evil invasion,belonging to the scope of Wen Disease.Chinese medicine Xingnaojing injection(Xingnaojing,XNJ)is from the "Treatise on differentiation and treatment of epidemic febrile disease",and is the modern improved preparations of ancient preparation of Angong Niuhuang pills.The major fuction of which is tepidity evil invaginate heart,phlegm obscure head.Clinically,taking dizzy dumb language,high fever irritability,red tongue or purple as the rule of governance points,which belongs to Yingxue syndrom.Studies have shown that XNJ acting on the central nervous system,with lower blood lipids,improves the role of blood viscosity.XNJ treat with cardiovascular and cerebrovascular diseases,with the exact effect.The development of cerebral malaria in the early stage of the study group showed that the development of the adhesion of Plasmodium falciparum was closely related to the factors such as TNF-α,ICAM-1 and VCAM-1,and confirmed that the pathological damage caused by the small blood vessels is the important cause of cerebral malaria high mortality.While with the use of artemisinin drugs to kill the protozoa,finding drugs to lift the protozoa blocking,is the key of improving the rate of cerebral malaria treatment.Yingxue syndrom of warm disease is a clinically critical ill,accompanied by severe injury and dysfunction of tissues and organs.The existing study found that the essence of Yingxue syndrom related to microcirculation disturbunce and dfisseminated intravascular coagulation mediated by inflammatory mediator,eventually leading to irreversible tissueand organ damage and multiple organ failure.While the resulting pathological changes is partly similar with the pathogenesis of cerebral malaria.In recent years,with the immunology,blood biochemistry,microcirculation pathology and other new methods of use,there are deeper understanding for the risk of Yingxue syndrom.However,there are few reports on the pathology of Yingxue syndrom in cellular,sub-cellular and molecular level.Although there are many treatments and drugs in the treatment,the objective indexes of evaluating the curative effect are few reported.In this study,we have established the monkey cerebral malaria animal model,by Plasmodium Knowlesi intravenous injecting into the rhesus monkeys.After successful modeling,the monkeys were treated with artesunate or artesunate combined with Xingnaojing injection.Observing effect of the two groups of drugs on treatment with monkey cerebral malaria with Yingxue syndrom,with particular attention to Xingnaojing whether to remove the protozoa blocking.The levels of TNF-a,ICAM-1 and VCAM-1 were measured by ELISA.The levels of TNF-α,ICAM-1 and VCAM-1 mRNA were detected by RT-PCR before and after treatment.The expression of CD36,ICAM-1 and VCAM-1 in the brain and spleen of the brain was detected by immunohistochemical technique before and after treatment.The purpose of ELISA,RT-PCR and immunohistochemical test is to discuss the possible mechanism of relieving microvascular obstruction caused by PRBC sequestion,and to find objective indicator diagnosing Yingxue syndrom.And then using RNA-Seq sequencing technology,artesunate and artesunate combined with Xingnaojing treatment effect of monkey cerebral malaria were suquenced and data analyzed,to explore the related core genes,bridge genes and signaling pathways of microvascular occlusion.Which would lay the material basis for explaining the traditional Chinese medicine injections Xingnaojing dredging small blood vessel obstruction,and improving the treatment rate of cerebral malaria and explaing its potential treatment mechanism.Methods1.There were 16 common healthy rhesus monkeys with weight of 4.0±0.5kg,male and female on half.They were randomly divided into control group and treatment group.The control group(n = 4)and the treated group(n = 12)were infected with Plasmodium Knowlesi.After the successful modeling of monkey cerebral malaria with Yingxue syndrom,the 12 monkeys were randomly divided into three groups:model group,artesunate group and artesunate + Xingnaojing group,each group with 4 Animals,male and female on half.2.4 rhesus monkeys of Model group were infected with Plasmodium Knowlesi.,modeling monkey cerebral malaria with Yingxue syndrom,while 4 animals of the control group as a blank control.Parasite in the 40～42 ℃ warm water,and then rapid injecting which into the rhesus monkey in the saphenous vein.While the inoculation day recorded as DO.From the beginning of D1,the monkey blood were collected every day,thick or thin blood film were prepared,and erythrocyte infection rate were calculated under microscopic.At the same time,clinical scores were done,and whether there is ataxia,lethargy,coma,convulsions and other symptoms were also observed,until the diagnostic criteria in line with CM.When the untreated model group died,dissected and subjected to pathological sections prepared and scored.3.Artesunate group and artesunate + Xingnaojing group,each group of four animals,were administrated with artesunate injection and artesunate + Xingnaojing injection,and the efficacy was observated.Efficacy index included the average protozoa negative time,the average fever time,the average awake time and cure rate.After the success of the model,blood samples were collected once a day before and 1,2,3,4,5,6 and 7 days after administration,and blood and blood biochemical tests were performed.Data were processed using the STATA 11 statistical software package.All results were expressed as x±s,using independent sample t-test and one-way ANOVA.4.The control group,model group,artesunate group and artesunate + Xingnaojing group,each group of 4 animals,using double antibody sandwich method for ELISA detection.The levels of TNF-a,sICAM-1 and sVCAM-1 in monkey serum were measured on the 1st,3rd,5th and 7th day before and after administration.Data were processed using the STATA 11 statistical software package.All results were expressed as x±s,using independent sample t-test and one-way ANOVA.5.The control group,model group,artesunate group and artesunate + Xingnaojing group,each group of 2 animals,male and female on half.At the end of the drug treatment,the expression levels of TNF-α,ICAM-1 and VCAM-13 mRNA were detected by Syber Green fluorescence quantitative PCR from the brain and spleen of each rhesus monkey.6.The control group,model group,artesunate group and artesunate + Xingnaojing group,each group of 2 animals,male and female on half.The expression of CD36,ICAM-1 and VCAM-1 in the brain and spleen were detected by immunohistochemistry after collecting brain and spleen of each rhesus monkey.7.The control group,model group,artesunate group and artesunate + Xingnaojing group,each group of 2 animals,male and female on half.At the end of the drug treatment,brain tissue was collected from each rhesus monkey,then sequenced and analyzed by RNA-Seq.In the process of data analysis,the differentially expressed genes of different groups of rhesus monkeys were respectively compared with the model group and Artesunate +Xingnaojing group,as well as the Artesunate + Xingnaojing group and Artesunate group.Results1.Establishment of monkey CM model with Yingxue syndrom infected with Plasmodium Knowlesi.When the rhesus monkey erythrocyte infection density ≥ 10%,the clinical score ≥ 3 points,and the model group animals began to appear disturbance of consciousness,coma,capillaries appearing PRBC and parasite accumulation of blood vessels or other cerebral malaria symptoms,at the same time,typical symptoms of Yingxue syndrom in the Chinese medicine appeared,such as heat into the blood,blood-heat bleeding,coma,Yin-yang dissociation,etc.,which is diagnosed as monkey cerebral malaria with Yingxue syndrom.The levels of TNF-α,sICAM-1 and sVCAM-1 in the model group were significantly higher than those in the control group by ELISA test(P<0.05,P<0.01,p<0.01).Compared with the control group,the expression of TNF-a mRNA and ICAM-1 mRNA in the model group was up-regulated than that in the control groupby RT-PCR.Immunohistochemical analysis showed that CD36 and ICAM-1 were up-regulated in the brain and spleen of the monkey,compared with the control group,suggesting that the above factors could be used as diagnostic indicator of monkey cerebral malaria with Yingxue syndrome.2.The efficacy of artesunate(artesunate group,the same below),artesunate + Xingnaojing(combined administration group,the same below)2 groups treatment with monkey cerebral malaria was compared,finding that the cure rate of combined administration group(100%)was higher than that of artesunate group(75%).Meanwhile,the average awake time,average fever time and average protozoan of combined administration group were lower than that of artesunate group,but not statistically significant.Traditional Chinese medicine injection Xingnaojing with removing heat to cool blood and inducing resuscitation,may improve the treatment rate of cerebral malaria through dredging obstruction in the small blood vessels,and reducing tissue damage caused by obstruction.Which provides a new way of thinking for cerebral malaria treatment.3.The molecular mechanism of artesunate group and combination administration group in the treatment of monkey cerebral malaria was investigated.The results showed that the combination administration group significantly reduced the levels of TNF-α,sICAM-1 and sVCAM-1(P<0.01,p<0.05,p<0.05).Compared with the artesunate group,RT-PCR results showed that the expression level of TNF-a mRNA and ICAM-1mRNA of the combination administration group in brain tissue was significantly down-regulated,compared with the artesunate group.Meanwhile,compared with artesunate group,the combination administration group could significantly down-regulate CD36 and ICAM-1 expression level in the spleen and brain tissue,suggesting that the above factors can be used as an objective diagnosis indicator of TCM Yingxue syndrom.4.The RNA-Seq sequencing and data analysis of the Artesunate + Xingnaojing group VS Model group and the Artesunate + Xingnaojing group VS Artesunate group were studied by RNA-Seq.The results showed that,compared with the Model group,the Artesunate +Xingnaojing group mediated vascular endothelial growth factor production pathway,endocytosis pathway and cytokine-cytokine receptor interaction pathway,and so on,and GNGT2,GNAI2,DGKQ and AMPD3 is the core gene for monkey CM treatment biology process of Artesunate + Xingnaojing group;Compared with the Artesunate group,the combination therapy group mediated the cell surface signal pathway,the cellular response of interleukin-21,phospholipase C-activated G protein-coupled signaling pathway,etc.,and PLB1,IL21R,ALDH3A2,ANAPC7,ERBB3 is the core gene of Xingnaojingjing in the monkey CM treatment biology process.Which would lay the material basis for explaining the traditional Chinese medicine injections Xingnaojing dredging small blood vessel obstruction,and improving the treatment rate of cerebral malaria and explaing its potential treatment mechanism.Conclusions1.In this study,through rhesus monkey blood transmission of Plasmodium knowlesi,when the monkey erythrocyte infection rate reached or exceeded 10%,the monkeys appeared coma,irritability,hematuria and other Yingxue syndrom evidence,rhesus monkey cerebral malaria model infected with Plasmodium Knowlesi with Yingxue syndrom was established.2.The monkeys were randomly divided into two groups after appearing Yingxue syndrom,namely artesunate injection for the artesunate group,Xingnaojing combined with artesunate injection as the combination administration group.The cure rate of the combination group(100%)was higher than that of the artesunate group(75%),and the average awake time,average fever time and average time of the combination group were significantly higher than those in the artesunate group.The time of protozoan was shorter than that of artesunate group,but not statistically significant.Traditional Chinese medicine injection Xingnaojing may be through the small blood vessels to reduce obstruction caused by tissue damage and improve the treatment rate of cerebral malaria,which provides a new way of thinking.3.The levels of TNF-α,sICAM-1 and sVCAM-1 of the model in peripheral blood of monkey serum were increased,and the expression of TNF-α mRNA,ICAM-1 mRNA and CD36 of the model in brain tissue was higher,compared with the control group;the levels of TNF-a,sICAM-1 and sVCAM-1 of the combined administration group in monkey serum were significantly decreased,and the level of of TNF-α mRNA,ICAM-1 mRNA and CD36 expression in the brain were down-regulated,compared with the artesunate group,All of the above may be an important indicator for the diagnosis of monkey cerebral malaria.Using RNA-Seq sequencing technology,the combination of artesunate and Xingnaojing to improve the prognosis of monkey cerebral malaria retrograde sequencing and data analysis,it is found that compared with the artesunate group,the combination of drug group mediated cell surface receptor gene pathway,interleukin-21 cell response pathway,phospholipase C activation G protein coupled signal pathway and other biological processes.While PLB1,IL21R,ALDH3A2,ANAPC7,ERBB3 is the related core genes of combined administration group with monkey cerebral malaria biological process.Suggesting the core genes may be related to the objective index of diagnosis of Yingxue syndrome.