Effects Of Mannose On Erythrocytic Stage Of Plasmodium

Objectives: Malaria is a global parasitic infectious disease caused by Plasmodium infection.Hundreds of thousands of patients in Africa and Southeast Asia die from the disease every year,among which cerebral malaria is the most serious complication and the leading cause of death.At present,there is no effective vaccine against malaria,which mainly depends on the combination therapy of antimalarial drugs based on artemisinin.However,the resistance of malaria parasite to artemisinin has become a serious challenge for our country and even the whole world.Therefore,prolonging the time of administration or replacing the adjuvant drugs that have developed drug resistance in artemisinin combination therapy will become an important strategy for malaria treatment in the future.The rational use of existing antimalarial drugs and actively looking for new adjuvant antimalarial drugs have also become a top priority.Tumor cells have an increased demand for glucose,which is similar to that of red blood cells infected with Plasmodium.Studies have shown that D-mannose is an isomer of glucose,which can use glucose transporter(GLUT)to enter tumor cells and inhibit tumor growth by interfering with glucose metabolism,promoting tumor cell apoptosis and enhancing the effect of chemotherapeutic drugs.The parasite also ingests glucose through GLUT on the surface of red blood cells and hexose transporter(HT)on the plasma membrane of the parasite.Therefore,we speculate that mannose can compete with glucose through GLUT and HT to enter the parasite and inhibit the growth of the parasite by interfering with glucose metabolism.The main purpose of this study is to study whether D-mannose can inhibit the growth of Plasmodium in erythrocytic stage and its related mechanism,and to explore the preventive effect of D-mannose on cerebral malaria and its possible mechanism.Methods: 1.BALB/c mice were given 20% mannose solution 200 μl by intragastric administration,once every other day,and the drinking water was changed into 20% mannose solution.The body weight and food intake of mice were monitored every day.After 20 days,the serum of mice was taken to detect the related indexes of liver and kidney function,glutamic pyruvic transaminase(ALT),glutamic oxaloacetic transaminase(AST),total bilirubin(TBILC),urea and creatinine(CRE).2.A mouse model of malaria was established by injecting 1×104 red blood cells infected with Plasmodium berghei into the tail vein of BALB/c mice.The malaria mice were given 20% mannose solution 200 μl by intragastric administration,once every other day,and the drinking water was changed into 20% mannose solution.1~2 μl blood was taken from the tip of tail every day to make blood smear,and the parasitemia was observed and counted by microscope after Giemsa staining.3.The initial parasitemia of Plasmodium falciparum 3D7 cultured in vitro was 2%.Different concentrations of mannose solutions(0m M,20 m M,40 m M and 50 m M)were treated respectively.Blood smears were taken every day,and the parasitemia was counted after Giemsa staining.4.The red blood cells infected with Plasmodium berghei were isolated from the blood of Wistar rats by density gradient centrifugation and cultured in vitro in 24-well plate.1×107 i RBC/wells were added with different concentrations of mannose solution(0m M,10 m M,20 m M and 30 m M).After incubation for 1 hour,the content of ROS was detected by DCFH-DA staining and flow cytometry.5.The cerebral malaria model of C57BL/6 mice was established by intravenous injection of 1×104 Plasmodium berghei i RBC into the tail vein.Cerebral malaria usually occurred 5~12 days after infection.200 μl of 20% mannose solution was given to the stomach every other day after infection,and the drinking water was changed into 20% mannose solution,while the control group was given normal saline in the same way.The parasitemia was monitored every day,and the infected mice were scored according to the rapid evaluation of cerebral malaria,and the incidence of cerebral malaria was counted.For the mice that met the diagnostic criteria of cerebral malaria,the brain tissue sections were prepared,and the pathological changes such as cerebral hemorrhage were evaluated after HE staining.If there were no symptoms of cerebral malaria,the pathological changes of brain tissue were detected on the 13 th day after infection.To analyze the role of mannose in the prevention of cerebral malaria.6.For the above mice cerebral malaria model,the brain tissue was taken when cerebral malaria occurred in mice,and if no cerebral malaria occurred,samples were taken on the 13 th day after infection.After total RNA was extracted and reverse transcription,the expression levels of β-actin,parasites 18 s RNA,TNF-α and IFN-γ in brain tissue were detected by realtime PCR,and the effect of mannose on the level of inflammatory factors in brain tissue of mice with cerebral malaria was analyzed.Results: 1.High-dose mannose had no effect on the body weight and intake of normal mice,and did not damage the liver and kidney function of mice.2.High-dose mannose can significantly inhibit the growth of Plasmodium berghei in erythrocytic stage of infected mice.3.High-dose mannose can significantly inhibit the growth of Plasmodium falciparum in vitro.4.High-dose mannose significantly increased the level of ROS in erythrocytes infected with Plasmodium berghei and inhibited the growth of Plasmodium berghei by causing oxidative stress.5.High-dose mannose can prevent the occurrence of cerebral malaria and significantly inhibit the cerebral hemorrhage in infected mice.6.High-dose mannose had no significant effect on sequestration of Plasmodium in the brain tissue of infected mice,but it could inhibit the occurrence of cerebral malaria by inhibiting the expression of inflammatory factors TNF-α and IFN-γ in brain tissue.Conclusions: High-dose mannose can inhibit the growth of Plasmodium in vivo and in vitro,which maybe due to the increase of ROS level in the parasite induced by mannose inhibited pentose phosphate pathway,which kills the parasite by oxidative stress.In addition,although high-dose mannose could not reduce sequestration of Plasmodium in the brain tissue of malaria-infected mice,it could significantly inhibit the expression of inflammatory factors in brain tissue,thus reducing the incidence of cerebral malaria.