Application of ackermania muciniphila in improving and treating cerebral malaria caused by plasmodium berghei

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of Ackermania muciniphila in improvement and treatment of cerebral malaria caused by Plasmodium berghei. The invention tries to evaluate the effect of the AKK bacterium on improving and treating the cerebral malaria of a mouse under the condition that the AKK bacterium or auxiliary medicines (dihydroartemisinin, rapamycin and atorvastatin) are independently used on a cerebral malaria model of the mouse. Results indicate that the strain can effectively improve behavioristics of mice and immune pathological injuries of all organs, reduce protozoemia and prolong the lifetime of the mice whether the strain is independently used or combined with the drugs, and compared with the strain which is independently used with the drugs, the strain can effectively treat cerebral malaria of the mice. As ackermania muciniphila is common probiotics in human intestinal tracts, the three drugs (dihydroartemisinin, rapamycin and atorvastatin) used in the invention are all clinically used drugs, and if the drugs are clinically transformed to treat human brain malaria caused by plasmodium falciparum, the feasibility is high, and meanwhile, the transformation time can be shortened. According to the invention, a bacterium-medicine synergistic concept is provided for the first time, that is, probiotics can assist medicines in treating diseases, and a solution is provided for treating human cerebral malaria under the guidance of the concept.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of Akkermansia muciniphila in improving and treating cerebral malaria in mice caused by Plasmodium berghei. Background Art

[0002] Malaria remains one of the most serious infectious diseases in the world. The 2021 World Health Organization report shows that the number of malaria cases has risen to 247 million and the number of deaths has risen to 619,000, a slight decrease compared to 2020. Cerebral malaria (CM) caused by Plasmodium falciparum is a fatal complication of malaria and the most common and serious parasitic disease affecting the human central nervous system. Artemisinin (ART) and its derivatives are currently the first-line treatment for CM. However, with the long-term use of antimalarial drugs, under the action of drug pressure, antimalarial drug resistance continues to increase. At the same time, the main function of antimalarial drugs is to eliminate malarial parasites in the body. The use of antimalarial drugs alone cannot comprehensively and effectively improve the neurological and brain pathological symptoms caused by immune pathological damage. Therefore, it is also necessary to explore new treatment options to assist drug treatment, make up for the shortcomings of antimalarial drugs, and thus treat cerebral malaria more comprehensively. At present, cerebral malaria in mice caused by Plasmodium berghei infection is the most commonly used animal model for studying human cerebral malaria.

[0003] Akkermansia muciniphila (Akk bacteria) is a normal bacterium in the human intestine and a mucin-degrading bacterium. The strain MucT was isolated by Derrien in 2004. It is an oval, Gram-negative anaerobic bacterium and a representative of the Verrucomicrobia. A. muciniphila is negatively correlated with obesity, diabetes, cardiovascular disease and low-grade inflammation. Oral administration of Akkermansia muciniphila can improve the symptoms related to metabolic diseases in mice, and Akkermansia muciniphila is expected to become a candidate drug for the treatment of type 2 diabetes and obesity. Intervention with this bacterium can prevent and treat obesity, ALS, enteritis, intestinal cancer and many other diseases.

[0004] At present, there are no reports on the improvement and treatment of cerebral malaria by A. muciniphila.

[0005] Drug treatment remains the first-line treatment for cerebral malaria (CM). However, decades of monotherapy have led to resistance to several recommended antimalarial drugs. Artemisinin (ART) and its derivatives are currently the first-line antimalarial drugs for the treatment of CM in adults, children and pregnant women. However, treatment with antimalarial drugs alone does not fully improve neurological and brain pathological damage, which reduces their effectiveness. In the absence of adjuvant therapy, the mortality rate of CM patients continues to rise. Therefore, inspired by the concept of combined treatment, it is urgent to explore combined treatments with ART that have an additive effect to reduce or prevent the occurrence of CM. In our previous study, we used a combination strategy of dihydroartemisinin combined with rapamycin and atorvastatin to treat cerebral malaria in mice. The results showed that compared with the use of dihydroartemisinin alone, it could effectively prolong the survival of mice and significantly alleviate immunopathological damage. However, the mechanism behind this phenomenon is unclear. In addition, we previously observed significant changes in the intestinal flora of mice after infection with different species of Plasmodium, which suggests that the intestinal flora may play an important role. Subsequently, based on the combination of the three drugs, we started from the intestinal flora and observed that compared with the use of dihydroartemisinin alone, the abundance of Akkermansia muciniphila (AKK bacteria for short) in the intestines of mice in the three-drug combination group was significantly increased, suggesting that this bacterium plays an important role in it.

[0006] Studies have shown that AKK bacteria play an important role in intestinal inflammatory diseases, tumors, nervous system diseases, etc. However, there have been no reports on infectious diseases, especially cerebral malaria. To this end, the present invention attempts to reveal the role of AKK bacteria in the treatment of mouse cerebral malaria and its possible mechanism of action. Specifically, a mouse cerebral malaria model was first established, and then AKK bacteria were used in combination with drugs or alone for treatment on the model, and the therapeutic effect of the bacteria on mouse cerebral malaria was observed by technical means such as behavior, changes in protozoaemia and pathology. Summary of the invention

[0007] The present invention first provides the use of Akkermansia muciniphila in improving and treating cerebral malaria caused by Plasmodium berghei, wherein the use is in vivo or in vitro; optionally, the improvement and treatment comprises one or more of the following:

[0008] A) Improve mouse behavior;

[0009] B) Improve the immune pathological damage of various organs;

[0010] C) reduce parasitemia;

[0011] D) prolong the survival of mice.

[0012] In certain embodiments, the in vitro is a bacterium.

[0013] In certain embodiments, the in vivo is a mouse.

[0014] In certain embodiments, the Akkermansia muciniphila is Akkermansia muciniphila ATCCBAA-835.

[0015] The invention also provides the use of Akkermansia muciniphila in preparing medicines for improving and treating cerebral malaria caused by Plasmodium berghei.

[0016] In certain embodiments, the drug further comprises one or three of dihydroartemisinin, rapamycin, and atorvastatin, wherein dihydroartemisinin is an essential drug.

[0017] In certain embodiments, the medicament further comprises dihydroartemisinin.

[0018] In certain embodiments, the medicament further comprises dihydroartemisinin and rapamycin.

[0019] In certain embodiments, the medicament further comprises dihydroartemisinin, rapamycin, and atorvastatin.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] Our previous results showed that AKK bacteria were significantly enriched in the three-drug combination group, suggesting that it may play a role in the treatment of mouse cerebral malaria. The present invention attempts to evaluate the effect of improving and treating mouse cerebral malaria in a mouse cerebral malaria model using AKK bacteria alone or auxiliary drugs (dihydroartemisinin, rapamycin, atorvastatin). The results suggest that whether the bacteria are used alone or in combination with drugs, they can effectively improve mouse behavior, immune pathological damage to various organs, reduce parasitemia, and prolong mouse survival. Compared with the use of drugs alone, they can effectively treat mouse cerebral malaria. Since the three drugs used (dihydroartemisinin, rapamycin, and atorvastatin) are all clinically used drugs, if they are clinically transformed for the treatment of human cerebral malaria, the conversion time can be shortened. The present invention proposes for the first time the concept of bacterial-drug synergy, and provides a solution for the treatment of human cerebral malaria under the guidance of this concept. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Morphological observation of Akkermansia muciniphila. A. Morphological image under optical microscope; B. Morphological image under scanning electron microscope.

[0023] Figure 2 Three-view observation of normal mice and mice with ECM (experimental cerebral malaria) symptoms.

[0024] Figure 3 Effects of different drug treatments on behavioral indicators of ECM mice. A. Body weight; B. RMCBS; C. Survival rate; D. Protozoaemia.

[0025] Figure 4 Effects of different drug treatments on mucosal barrier function in ECM mice. A. Frontal view of brain tissue; B. EB test to evaluate vascular leakage; C. Intestinal permeability.

[0026] Figure 5 Histopathological observation of HE staining of mice treated with different drugs. A. Brain tissue (1000×); B. Liver (400×); C. Spleen (400×); D. Small intestine (400×). DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0028] Abbreviations:

[0029] DHA: dihydroartemisinin;

[0030] RAP: rapamycin;

[0031] AVA: atorvastatin;

[0032] Example 1 Materials and Methods

[0033] Experimental animals, Plasmodium strains and strains: Female C57BL / 6 mice (weight 18-22 g, 8-10 weeks old) were purchased from HNSJA Co., Ltd., Changsha, China. They were raised under specific pathogen-free conditions and fed with UV-irradiated feed and purified water to maintain appropriate living and feeding standard experimental conditions (25±3℃). All mice were acclimatized to the environment 1 week before the experiment.

[0034] Plasmodium berghei ANKA was kindly donated by Professor Xu Wenyue of the Army Medical University and was cryopreserved in liquid nitrogen for long-term storage in our laboratory. Akkermansia muciniphila ATCC BAA-835 was purchased from ATCC (http: / / www.bncc.org.cn / pro / p75 / p_323275.html).

[0035] The experimental instruments are shown in Table 1;

[0036] Table 1 Experimental instruments

[0037]

[0038]

[0039] Experimental reagents and consumables are shown in Table 2;

[0040] Table 2 Experimental reagents and kits

[0041] name Manufacturer Giemsa stain China, Rain Dihydroartemisinin China, Myrel Rapamycin China, Myrel Atorvastatin Solarbio, China syringe China, Weigao Cryotubes Thermo Fisher Scientific, USA Glass slides China, Shitai Methanol China, Zhongtian Dimethyl sulfoxide VWR, USA EvansBlue SIGMA, Germany Formamide SIGMA, Germany 0.9% saline Kelun, China Universal tissue fixative China, Absin Slide Storage Box China, Blue Sky PBS buffer powder Servicebio, UK Pipette Tips Thermo Fisher Scientific, USA

[0042] Example 2

[0043] Resuscitation and passage: Place the PbA malarial parasite strain frozen in liquid nitrogen in a 37°C water bath, and draw blood with a 1ml syringe after it is thawed. Immediately inoculate it intraperitoneally into C57BL / 6 mice at a dose of 0.2mL / mouse. This is blood seed resuscitation. When the parasitemia level of the blood seed mice reaches 15%-30%, remove the eyeballs and draw blood in a heparin anticoagulant tube. 6 The iRBCs-infected erythrocytes were passaged into offspring C57BL / 6 mice at a dose of 0.2 mL / mouse.

[0044] (1) AKK bacterial culture and extracellular vesicle preparation

[0045] A. muciniphila culture: culture medium (3.85 g brain heart infusion, 1.6 g soy peptone, 1.13 g anhydrous glucose, 0.55 g N-acetylglucosamine, 0.4 g L-threonine, 0.05 g L-cysteine, 100 mL) was used for anaerobically culture at 37°C for 3 days until the bacterial solution became slightly turbid. After counting, the bacterial precipitate was collected by centrifugation, washed, and resuspended in PBS for later use.

[0046] (2) Experimental groups and drug treatment

[0047] Sixty female C57BL / 6 mice were randomly divided into 6 groups: infection-untreated group (PbA), DHA-treated group (DHA), DHA combined with RAP and AVA-treated group (DHA.RAP.AVA), PbA+AKK group, DHA+AKK group, and DHA.RAP.AVA+AKK group.

[0048] DHA, RAP and AVA were dissolved in 5% DMSO and 0.9% NaCl solution at 3 mg / kg, 5 mg / kg and 40 mg / kg, respectively. On the 3rd day after infection, the PbA group was intraperitoneally injected with 5% DMSO as a control. The DHA group was given 3 mg / kg DHA. The DHA.RAP.AVA group was given 5 mg / kg RAP, 3 mg / kg DHA and 40 mg / kg AVA. The three groups containing AKK (PbA+AKK group, DHA+AKK group and DHA.RAP.AVA+AKK group) were gavaged with 200 μL of 10 9cfu / mLAKK bacterial solution, 3mg / kg DHA, 3mg / kg DHA+5mg / kg RAP+40mg / kg AVA, 200μL / d every day for 5 consecutive days after the third day of infection.

[0049] (3) Basic indicator testing

[0050] Protozoaemia observation: Blood was collected from the tail vein to prepare thin blood smears, which were then stained with Giemsa. Protozoaemia was evaluated by Giemsa-stained thin blood smears under an optical microscope with a magnification of 10x and an objective of 100x. Protozoaemia was examined and quantified by counting the number of iRBCs in at least 1,000 red blood cells.

[0051] (4) Detection of basic behavioral indicators: Starting from day 0 after infection, the weight, neurological characteristics, parasitemia and survival rate of mice were monitored daily. The rapid murine coma and behavioral scale (RMCBS) was used to evaluate neurological characteristics, and 10 parameters (hair, limb strength, defense ability, auricular reflex, toe reflex, contact reflex, gait, balance, body posture and exploratory activity) were used for evaluation. If mice gradually develop characteristics such as unsteady walking, ataxia, messy hair, no stretching, loss of toe reflex, loss of auricular reflex, convulsion, coma, and even death, ECM was successfully established. Parasitemia was evaluated and quantified by counting the number of iRBCs in 1,000 red blood cells by thin blood film smear method under a 1000× optical microscope.

[0052] (5) Assessment of blood-brain barrier integrity: The protective effect on the brain was assessed by the integrity of the blood-brain barrier (BBB). 1% EB dye was diluted in 0.9% NaCl. On the 8th day after infection, 200 μL of 1% EB dye solution was injected into the tail vein of the mouse, and the dye was allowed to circulate for 30 minutes. The mouse was then anesthetized and 0.9% NaCl was perfused into the right atrium through the heart to allow the clear liquid to flow out. The brain was quickly separated, photographed, weighed, and then placed in a 1.5 mL centrifuge tube for grinding. 1 mL of formamide was added to each brain sample and then incubated in a constant temperature metal bath at 37°C for 48 hours. The samples were centrifuged at 1000 rpm for 10 minutes, and the absorbance was measured at 630 nm using an enzyme reader. EB was quantified according to the standard curve.

[0053] (6) Histopathological observation: After the end of drug administration, i.e., on the 8th day after infection, tissue samples were collected. After euthanasia, brain, liver, spleen, and small intestine tissues were immediately collected and washed three times in cold PBS to remove blood. Then, they were fixed with a universal tissue fixative for 24 hours and embedded in paraffin. Continuous 4 μm thick sections were cut and HE stained to observe microvascular obstruction and leakage. The sections were observed using an optical microscope, and images were acquired using Olympus cellSens standard 1.13 software.

[0054] result:

[0055] (1) For the morphology of AKK under the microscope, see Figure 1 ;

[0056] (2) Successful establishment of ECM model: The animal model of cerebral malaria caused by PbA infection in C57BL / 6 mice was established. 6 Infected red blood cells were injected intraperitoneally into C57BL / 6 mice, which lost weight and began to die after 6-8 days. RMCBS scores showed ECM symptoms, and blood samples were taken from the tail of the mice to reveal anemia, indicating that the ECM model was successfully established (see Figure 2 ).

[0057] (3) Evaluation of basic indicators: The basic behavioral indicators showed that the DHA+RAP+AVA+AKK treatment group had the highest body weight, RMCBS score and survival rate ( Figure 3 AC), while the lowest level of parasitemia ( Figure 3 D), illustrating that AKK bacteria may play a synergistic role in the combined bacterial and drug treatment of ECM, see Table 3.

[0058] Table 3. Basic index average value record table

[0059]

[0060]

[0061] Note: The time selected in the table is based on the data of the PbA+AKK group as a benchmark reference, where D8, D9, D14 and D16 represent the days after infection.

[0062] Compared with the PbA+AKK group (AKK intervention in the mouse cerebral malaria model) and the DHA group (DHA treatment in the mouse cerebral malaria model), the average weight of the mice in the DHA+AKK group was 31.97% heavier than that in the PbA+AKK group and the DHA group on day 14 [(DHA+AKK group - PbA+AKK group) / PbA+AKK group] and 3.47% heavier [(DHA+AKK group - DHA group) / DHA group]. On day 9, the RMCBS scores of the mice in the DHA+AKK group were increased by 422.68% [(DHA+AKK group - PbA+AKK group) / PbA+AKK group] and 4.77% [(DHA+AKK group - DHA group) / DHA group] compared with those in the PbA+AKK group and the DHA group, respectively. On the 8th day, the parasitemia of mice in the DHA+AKK group was reduced by 91.21% [(DHA+AKK group - PbA+AKK group) / PbA+AKK group] and 27.21% [(DHA+AKK group - DHA group) / DHA group] compared with the PbA+AKK group and DHA group, respectively.

[0063] Compared with the PbA+AKK group (only AKK intervention was used in the mouse cerebral malaria model) and the DHA+RAP+AVA group (only DHA+RAP+AVA treatment was used in the mouse cerebral malaria model), on the 14th day, the average body weight of mice in the DHA+RAP+AVA+AKK group was 35.18% [(DHA+RAP+AVA+AKK group - PbA+AKK group) / PbA+AKK group] and 6.04% [(DHA+RAP+AVA+AKK group - DHA group) / DHA group] heavier than those in the PbA+AKK group and the DHA+RAP+AVA group, respectively. On the 9th day, the RMCBS scores of mice in the DHA+RAP+AVA+AKK group increased by 475.6% [(DHA+RAP+AVA+AKK group-PbA+AKK group) / PbA+AKK group] and 15.38% [(DHA+RAP+AVA+AKK group-DHA group) / DHA group] compared with those in the PbA+AKK group and the DHA group, respectively. On the 8th day, the parasitemia of mice in the DHA+RAP+AVA+AKK group decreased by 97.47% [(DHA+RAP+AVA+AKK group-PbA+AKK group) / PbA+AKK group] and 79.04% [(DHA+RAP+AVA+AKK group-DHA group) / DHA group] compared with those in the PbA+AKK group and the DHA group, respectively.

[0064] (4) Blood-brain barrier and intestinal permeability: The results of the BBB permeability assessment experiment showed that the brain tissue of mice in the DHA+RAP+AVA+AKK group had the lightest staining ( Figure 4 A), and the EB leakage was the lowest ( Figure 4 B); Intestinal permeability test showed that the OD value of AKK group was lower ( Figure 4 C). The above results suggest that the mucosal barrier function of mice was enhanced after AKK-assisted treatment, and the blood-brain barrier permeability and intestinal permeability were improved.

[0065] (5) Organ pathological analysis: Histopathological staining of brain, liver and spleen tissues. Figure 5 A Brain tissue: blue arrows point to red blood cells (RBCs), red arrows point to brain microvascular endothelial cells (BMECs), and the yellow oval box is the rosette effect. Figure 5 B Liver: The green rectangle is hemochromatin, and the blue oval is the adhesion of leukocytes and iRBCs. Figure 5 C Spleen: The red area represents the red pulp (RePu) and the blue area represents the white pulp (WhPu). Figure 5 D Small intestine: The purple arrow indicates the destruction and loss of goblet cells in the small intestinal villi, the blue arrow indicates the separation of intestinal epithelial cells, and the purple oval indicates the sequestration and bleeding of red blood cells in the blood vessels.

[0066] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of Akkermansia muciniphila in improving and treating cerebral malaria caused by Plasmodium berghei, characterized in that: The application is in vivo or in vitro; Optionally, the improvement and treatment comprises one or more of the following: A) Improve the behavioral performance of mice; B) Improve the immune pathological damage of various organs; C) reduce parasitemia; D) prolong the survival of mice.

2. The use according to claim 1, characterized in that: The in vivo is a mouse, and the in vitro is a bacterium.

3. The use according to claim 1, characterized in that: The Akkermansia muciniphila is Akkermansia muciniphila ATCC BAA-835.

4. The use of Akkermansia muciniphila in the preparation of drugs for improving and treating cerebral malaria caused by Plasmodium berghei.

5. The use according to claim 4, characterized in that: The drug also contains one or three of dihydroartemisinin, rapamycin, and atorvastatin, among which dihydroartemisinin is the essential drug.

6. The use according to claim 4, characterized in that: The medicament also contains dihydroartemisinin.

7. The use according to claim 4, characterized in that: The drug also contains dihydroartemisinin and rapamycin.

8. The use according to claim 4, characterized in that: The drug also contains dihydroartemisinin, rapamycin and atorvastatin.

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