Application of Akkermansia muciniphila in improving and treating cerebral malaria caused by Plasmodium berghei

Abstract

The present invention belongs to the field of biomedical technology and specifically relates to the use of Akkermansia muciniphila in ameliorating and treating cerebral malaria caused by Plasmodium berghei. This study evaluated the efficacy of Akkermansia muciniphila alone or in combination with adjuvant drugs (dihydroartemisinin, rapamycin, and atorvastatin) in ameliorating and treating cerebral malaria in a mouse cerebral malaria model. The results suggest that the bacteria, whether used alone or in combination with other drugs, effectively improved mouse behavior, immune pathological damage to various organs, reduced parasitemia, and prolonged mouse survival, demonstrating its effectiveness in treating cerebral malaria in mice compared to using the drugs alone. Because Akkermansia muciniphila is a common probiotic in the human gut, the three drugs used in this study (dihydroartemisinin, rapamycin, and atorvastatin) are all currently in clinical use. Clinical translation for the treatment of human cerebral malaria caused by Plasmodium falciparum is highly feasible and can also shorten the conversion time. This study proposes for the first time the concept of bacterial-drug synergy, whereby probiotics can complement drug treatments, and provides a solution for the treatment of human cerebral malaria based on this concept.

Description

Technical Field

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

[0002] Malaria remains one of the world's most serious infectious diseases. A 2021 World Health Organization report showed that the number of malaria cases rose to 247 million, and the number of deaths rose 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 severe 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, drug resistance is increasing under the influence of drug pressure. Furthermore, the primary function of antimalarial drugs is to eliminate Plasmodium parasites from 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, new treatment options need to be explored to assist drug treatment, compensate for the shortcomings of antimalarial drugs, and thus achieve a more comprehensive treatment of cerebral malaria. Currently, cerebral malaria in mice caused by Plasmodium berghei infection is the most commonly used animal model for studying human cerebral malaria.

[0003] Akkermansia muciniphila (Akkermansia muciniphila) is a normal intestinal bacterium that decomposes mucins. Strain MucT, isolated by Derrien in 2004, is an oval, Gram-negative anaerobic bacterium and a representative of the Verrucomicrobia phylum. A. muciniphila is negatively correlated with obesity, diabetes, cardiovascular disease, and low-grade inflammation. Oral administration of A. muciniphila improves symptoms associated with metabolic diseases in mice, making it a promising candidate for the treatment of type 2 diabetes and obesity. Intervention with this bacterium has been shown to prevent and treat a variety of conditions, including obesity, amyotrophic lateral sclerosis, enteritis, and colorectal cancer.

[0004] Currently, there are no reports on the use of A. muciniphila to improve and treat cerebral malaria.

[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 treating CM in adults, children, and during pregnancy. However, treatment with antimalarial drugs alone does not fully ameliorate neurological and brain pathology, which reduces their effectiveness. In the absence of adjunctive therapy, the mortality rate of patients with CM continues to rise. Therefore, inspired by the concept of combination therapy, there is an urgent need to explore combination therapies that have additive effects with ART to reduce or prevent the development of CM. We previously used a combination strategy of dihydroartemisinin, rapamycin, and atorvastatin to treat cerebral malaria in mice. Compared with dihydroartemisinin alone, the combination strategy effectively prolonged mouse survival and significantly alleviated immunopathological damage. However, the underlying mechanism of this phenomenon remains unclear. In addition, we have previously observed significant changes in the intestinal microbiota of mice infected with different Plasmodium species, suggesting that the intestinal microbiota may play a key 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, and neurological diseases. 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 cerebral malaria in mice 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. The therapeutic effect of the bacteria on mouse cerebral malaria was observed through technical means such as behavioral, parasitemia changes, 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 immune pathological damage to 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 present 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 the 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 by using AKK bacteria alone or auxiliary drugs (dihydroartemisinin, rapamycin, atorvastatin) in a mouse cerebral malaria model. The results suggest that whether used alone or in combination with drugs, the bacteria can effectively improve mouse behavior, immune pathological damage to various organs, reduce parasitemia, and prolong mouse survival. Compared with the use of drugs alone, it can effectively treat mouse cerebral malaria. Since the three drugs used (dihydroartemisinin, rapamycin, 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 and 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 an optical microscope; B. Morphological image under a 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 the mucosal barrier function of ECM mice. A. Frontal view of brain tissue; B. Vascular leakage assessed by EB assay; C. Intestinal permeability.

[0026] Figure 5 Histopathological observation of mice treated with different drugs by HE staining. 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 clearer, 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 Insect Strains: Female C57BL / 6 mice (weighing 18–22 g, 8–10 weeks old) were purchased from HNSJA Co., Ltd., Changsha, China. Mice were housed under specific pathogen-free conditions and fed a UV-irradiated diet and purified water to maintain appropriate living and feeding conditions (25 ± 3°C). All mice were acclimated to the environment 1 week prior to the experiment.

[0034] Plasmodium berghei strain ANKA was kindly provided by Professor Xu Wenyue of the Army Medical University and was subcultured and cryopreserved in liquid nitrogen 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 and Dew Dihydroartemisinin China, Myrel Rapamycin China, Myrel Atorvastatin Solarbio, China syringe Weigao, China Cryogenic tubes Thermo Fisher Scientific, USA slides China, Shitai Methanol China, Zhongtian dimethyl sulfoxide VWR, USA Evans Blue SIGMA, Germany Formamide SIGMA, Germany 0.9% saline Kelun, China Universal tissue fixative China, Absin Slide storage box China, Blue Sky PBS buffer powder UK, Servicebio Pipette tips Thermo Fisher Scientific, USA

[0042] Example 2

[0043] Resuscitation and passage: Place the liquid nitrogen-frozen PbA parasite strain in a 37°C water bath. After thawing, draw blood with a 1ml syringe and 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 collect 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) anaerobically at 37°C for 3 days until the culture becomes slightly turbid. Count and collect the bacterial pellet by centrifugation, wash, and resuspend in PBS for later use.

[0046] (2) Experimental groups and drug treatment

[0047] Sixty female C57BL / 6 mice were randomly divided into six 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 drugs. 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 starting from the 3rd day after infection.

[0049] (3) Basic indicator testing

[0050] Prostozoaemia: Blood was collected from the tail vein and thin blood smears were prepared and stained with Giemsa. Prostozoaemia was assessed on these thin blood smears under a light microscope at 10x magnification and 100x magnification. Prostozoaemia was detected and quantified by counting the number of iRBCs in at least 1,000 red blood cells (RBCs).

[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. Neurological characteristics were evaluated using the rapid murine coma and behavioral scale (RMCBS), which uses 10 parameters (hair, limb strength, defense ability, auricular reflex, toe reflex, touch reflex, gait, balance, body posture and exploratory activity) for evaluation. If mice gradually develop characteristics such as unsteady walking, ataxia, messy hair, no stretching, loss of toe reflex, loss of auricular reflex, convulsions, coma, or even death, ECM is successfully established. Parasitemia is evaluated and quantified by counting the number of iRBCs in 1,000 red blood cells by thin blood film smear under a 1000× optical microscope.

[0052] (5) Assessment of blood-brain barrier integrity: The protective effect on the brain was assessed by blood-brain barrier integrity (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 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. These samples were centrifuged at 1000 rpm for 10 minutes, and the absorbance was measured at 630 nm using a microplate reader. EB was quantified according to the standard curve.

[0053] (6) Histopathological observation: Tissue samples were collected after the end of drug administration, that is, on the 8th day after infection. 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. Serial 4 μm thick sections were cut and stained with HE 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 morphological diagram under AKK 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, and the mice lost weight and began to die after 6-8 days. The RMCBS score showed ECM symptoms, and blood samples were taken from the tail of the mice and anemia was found, 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 indicator 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), the DHA+AKK group (AKK intervention in the mouse cerebral malaria model) and the DHA group (DHA treatment in the mouse cerebral malaria model) showed that on day 14, the average body weight of the mice in the DHA+AKK group was 31.97% heavier [(DHA+AKK group - PbA+AKK group) / PbA+AKK group] and 3.47% heavier [(DHA+AKK group - DHA group) / DHA group] than those in the PbA+AKK group and the DHA group, respectively. 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), the DHA+RAP+AVA+AKK group (only DHA+RAP+AVA treatment 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 day 9, 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 and DHA groups, respectively. On day 8, parasitemia in 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 and DHA groups, 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). These results suggest that AKK-assisted treatment enhanced the mucosal barrier function of mice, and improved both blood-brain barrier permeability and intestinal permeability.

[0065] (5) Organ pathological analysis: Histopathological staining of brain, liver, and spleen tissues. Figure 5 A Brain tissue: blue arrows indicate red blood cells (RBCs), red arrows indicate brain microvascular endothelial cells (BMECs), and the yellow oval box indicates the rosette effect. Figure 5 B Liver: The green rectangle is hemochromatin, and the blue oval circle 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 within the scope of protection of the present invention.

Claims

1. The use of Akkermansia muciniphila in the preparation of drugs for improving and treating cerebral malaria caused by Plasmodium berghei; The improvement and treatment include one or more of the following: A) Improved behavioral performance of mice; B) Improve immune pathological damage to various organs; C) reduce parasitemia; D) prolong the survival of mice; The Akkermansia muciniphila is Akkermansiamuciniphila ATCC BAA-835.

2. The use according to claim 1, characterized in that The drug also contains one or three of dihydroartemisinin, rapamycin, and atorvastatin, among which dihydroartemisinin is the essential drug.

3. The use according to claim 1, characterized in that The drug also contains dihydroartemisinin.

4. The use according to claim 1, characterized in that The drug also contains dihydroartemisinin and rapamycin.

5. The use according to claim 1, characterized in that The drug also contains dihydroartemisinin, rapamycin and atorvastatin.

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