AKK and / or outer membrane vesicles thereof induce macrophage polarization method and application in preparation of drugs for promoting macrophage phagocytosis of infected red blood cells

Abstract

The application discloses an AKK and / or outer membrane vesicle thereof induced macrophage polarization method and application thereof in preparation of a medicine for promoting phagocytosis of infected red blood cells by macrophages, and belongs to the technical field of biological medicines. It is found by the application that macrophages are subjected to M1 polarization phenomenon after being induced by AKK bacteria or OMVs thereof. The macrophages subjected to M1 polarization produce proinflammatory factors, and phagocytose more red blood cells infected by Plasmodium. In the absence of existing anti-malaria medicines, compared with macrophages not subjected to M1 polarization, the macrophages subjected to M1 polarization only by AKK bacteria or AKK-OMVs can phagocytose more red blood cells infected by Plasmodium, effectively avoiding the resistance of Plasmodium to anti-malaria medicines caused by long-term use of the anti-malaria medicines, and providing a new scheme for clinical malaria treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for inducing macrophage polarization using Akkermansia muciniphila (AKK) and / or its outer membrane vesicles, and its application in the preparation of drugs that promote macrophage phagocytosis of infected erythrocytes. Background Technology

[0002] Malaria is one of the three major global public health problems posing a serious threat to human health. Antimalarial drugs, including quinine, chloroquine, mefloquine, sulfadoxine / pyrimethamine, artemisinin, and piperaquine, are the first-line treatment for malaria. However, long-term use of antimalarial drugs has led to mutations in the genes of the malaria parasite, resulting in drug resistance and significantly reducing the effectiveness of antimalarial drugs. With the increasing problem of drug resistance, researchers are exploring new antimalarial drugs. For example, some novel artemisinin derivatives and non-artemisinin drugs are undergoing laboratory research, preclinical studies, and clinical trials. The development of these compound drugs is lengthy, costly, and has a very low success rate. Based on the experience of malaria parasites developing resistance to previously used first-line antimalarial drugs (quinine, chloroquine, mefloquine, sulfadoxine / pyrimethamine) and current first-line antimalarial drugs (artemisinin), even if new drugs are successfully developed for malaria treatment, drug resistance will likely continue in the foreseeable future due to the genetic mutations caused by the compounds in the parasite under drug stress. Therefore, bypassing the development of new compounds as antimalarial drugs is a new way to avoid drug resistance in malaria parasites.

[0003] Prior art CN119367402A discloses the application of *Akkermansia myxophilus* in improving and treating cerebral malaria caused by *Plasmodium berghei*. In a mouse model of cerebral malaria, the efficacy of *Akkermansia myxophilus* alone or in combination with adjuvant drugs (dihydroartemisinin, rapamycin, atorvastatin) in improving and treating cerebral malaria was evaluated. Results indicate that, whether used alone or in combination with drugs, *Akkermansia myxophilus* effectively improves behavioral changes, immunopathological damage to various organs, reduces protozoemia, and prolongs the survival of mice. Compared with drug use alone, it is an effective treatment for cerebral malaria in mice.

[0004] Existing technology CN111840250B discloses a novel reagent for the treatment of malignant cerebral malaria. This reagent consists of a hollow protein shell formed by the self-assembly of 24 protein subunits and an iron-based nanozyme with catalase activity, which can specifically target brain microvascular endothelial cells and scavenge reactive oxygen species (ROS). First, because the protein shell of this reagent has iron ion channels, uniformly sized iron nanonuclei can be synthesized within its cavity. These iron nanonuclei possess catalase catalytic activity, enabling the decomposition of hydrogen peroxide. Second, the ferritin shell can target brain endothelial cells while simultaneously promoting the proliferation and M1 subtype polarization of macrophages in the liver, enhancing their phagocytic function against infected red blood cells. Therefore, this reagent can be used to treat cerebral malaria.

[0005] Prior art CN111918967B discloses compositions and methods comprising extracellular vesicles, wherein the extracellular vesicles contain nucleic acids that target genes and induce macrophage polarization in tumor-associated macrophages. It also discloses the use of extracellular vesicles in the preparation of compositions regulating gene expression in macrophages.

[0006] The prior art CN117264871A discloses the application of Artemisia annua exovesicles in drugs that regulate the immune microenvironment, using Artemisia annua exovesicles as immunomodulators and co-culturing them with macrophages to polarize macrophages from the M2-like phenotype to the M1 phenotype in vivo and in vitro.

[0007] Macrophages are among the main effector cells of the innate immune system and play a crucial role in innate immunity. Macrophages can clear pathogens through phagocytosis. Activated macrophages enhance this phagocytic activity. Therefore, improving the phagocytic activity of macrophages against erythrocytes infected with Plasmodium is an effective measure to address antimalarial drug resistance. Summary of the Invention

[0008] The purpose of this invention is to provide the use of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of medicaments for treating diseases caused by Plasmodium, thereby addressing the problems existing in the prior art. This invention discloses a method comprising inducing M1 polarization of macrophages with AKK bacteria and their outer membrane vesicles, resulting in polarized macrophages enhanced phagocytosis of erythrocytes infected with Plasmodium.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] One of the technical solutions of the present invention is a method for inducing macrophage polarization using Akkermansia muciniphila (AKK) and / or its outer membrane vesicles, the method comprising the step of inducing macrophage polarization using Akkermansia muciniphila (AKK) and / or its outer membrane vesicles.

[0011] In some embodiments, the polarization is a transition from an unpolarized M0 state to a polarized M1 state or a reversal from a polarized M2 state to a polarized M1 state.

[0012] In some embodiments, the Akkermansia muciniphila is Akkermansia muciniphila JCM 33894T strain, purchased from the Japan Microbial Collection (JCM).

[0013] In some embodiments, the M1-polarized macrophages produce pro-inflammatory factors and simultaneously engulf more malaria-infected red blood cells.

[0014] The second technical solution of the present invention is an M1 polarized macrophage, wherein the M1 polarized macrophage is prepared by the above-described method.

[0015] The third technical solution of the present invention is the application of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of a drug that promotes macrophage phagocytosis of infected erythrocytes.

[0016] The fourth technical solution of the present invention is the application of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of antimalarial drug-resistant compositions.

[0017] The fifth technical solution of the present invention is the use of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of a composition for regulating gene expression in macrophages.

[0018] The sixth technical solution of the present invention is the application of the above-mentioned M1 polarized macrophages in the preparation of antimalarial drug resistant compositions.

[0019] Based on the above technical solution, the present invention has the following technical effects:

[0020] This invention discovers that macrophages undergo M1 polarization after being induced by AKK bacteria or its OMVs. M1-polarized macrophages produce pro-inflammatory factors and simultaneously engulf more infected red blood cells. This invention enables the phagocytosis of Plasmodium-infected red blood cells solely through polarized macrophages without the use of drugs. This method effectively avoids the long-term use of antimalarial drugs, preventing Plasmodium from developing resistance to them. It eliminates the need for drug-based clearance of infected red blood cells by using phagocytosis to remove the pathogen, effectively preventing drug resistance. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Morphology of AKK bacteria and AKK-OMVs. Among them, (A) Gram staining image of AKK bacteria under light microscopy, scale bar: 10 μm; (B) Morphology of AKK bacteria under transmission electron microscopy (TEM), scale bar: 500 nm; (C) Morphology of AKK-OMVs under transmission electron microscopy, scale bar: 200 nm.

[0023] Figure 2 For observation of protozoemia.

[0024] Figure 3 To verify the effects of AKK and AKK-OMVs on macrophage polarization using RT-qPCR. In the figures, A represents the relative expression level of iNOS mRNA, B represents the relative expression level of CD86 mRNA, C represents the relative expression level of TNF-α mRNA, D represents the relative expression level of Arg-1 mRNA, E represents the relative expression level of CD206 mRNA, and F represents the relative expression level of TGF-β mRNA.

[0025] Figure 4 To verify the depolarization effect of AKK and AKK-OMVs on M2 polarized macrophages by RT-qPCR. In the figures, A represents the relative expression levels of iNOS mRNA, B represents the relative expression levels of CD86 mRNA, C represents the relative expression levels of TNF-α mRNA, D represents the relative expression levels of Arg-1 mRNA, E represents the relative expression levels of CD206 mRNA, and F represents the relative expression levels of TGF-β mRNA. G represents the mass spectrometry analysis results of AKK-OMVs components.

[0026] Figure 5 To observe the effects of AKK and AKK-OMVs on macrophage polarization by flow cytometry.

[0027] Figure 6 To observe the effect of AKK and AKK-OMVs on the depolarization of M2 macrophages by flow cytometry.

[0028] Figure 7Giemsa staining smears were used to observe the phagocytosis of infected erythrocytes (iRBCs) by macrophages co-incubated for 1 hour. A is the BLK group (blank group), B is the IFN-γ+LPS group (classical M1 polarization group), C is the IL-4 group (classical M2 polarization group), D is the AKK group, E is the AKK-OMVs group, and F shows the phagocytosis rate of infected erythrocytes by macrophages in each group.

[0029] Figure 8 To observe the phagocytosis of infected erythrocytes by macrophages after M1 polarization induced by AKK bacteria and AKK-OMVs under different microscopes. (AC) shows the phagocytosis of infected erythrocytes by macrophages after M1 polarization induced by AKK bacteria under light microscopy, scanning electron microscopy, and transmission electron microscopy, respectively; (DF) shows the phagocytosis of infected erythrocytes by macrophages after M1 polarization induced by AKK-OMVs under light microscopy, scanning electron microscopy, and transmission electron microscopy, respectively.

[0030] Figure 9 The results are for the macrophage clearance and replenishment experiment. Among them, A is the body weight statistics of mice in the BLK, NEG, M0, M1 and M2 groups from D0 to D7; B is the RMCBS score statistics of mice in the BLK, NEG, M0, M1 and M2 groups from D2 to D7; and C is the protozoemia statistics of mice in the BLK, NEG, M0, M1 and M2 groups from D0 to D6. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0037] This invention provides the use of Akkermansia muciniphila and / or its outer membrane vesicles in the preparation of medicaments for treating diseases caused by Plasmodium.

[0038] In some specific implementations, the Akkermansia muciniphila JCM 33894T strain was purchased from the Japan Microbial Collection (JCM).

[0039] In some specific implementations, the malaria parasite includes Plasmodium berghei ANKA.

[0040] In some specific implementations, Akkermansia muciniphila promotes the phagocytosis of malaria-infected red blood cells by stimulating M1 macrophage polarization.

[0041] This invention also provides a drug for treating diseases caused by Plasmodium, including Akkermansiamuciniphila and / or its outer membrane vesicles.

[0042] This invention also provides the use of Akkermansia muciniphila and / or its outer membrane vesicles in the preparation of drugs that promote macrophage M1 polarization.

[0043] This invention also provides the use of Akkermansia muciniphila and / or its outer membrane vesicles in the preparation of drugs that reverse macrophage M2 polarization.

[0044] In this invention, RBCs: normal red blood cells; iRBCs: red blood cells infected with Plasmodium, abbreviated as infected red blood cells or infected red blood cells.

[0045] Example 1

[0046] 1. Materials and Methods

[0047] Laboratory animals, Plasmodium strains and 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, fed ultraviolet-irradiated feed and purified water to maintain appropriate living and feeding standards (25±3℃). All mice underwent environmental acclimatization one week prior to the experiment.

[0048] The Plasmodium berghei ANKA (PbA) strain was kindly donated by Professor Xu Wenyue of the Army Medical University and has been cryopreserved in liquid nitrogen by our laboratory after passage.

[0049] Akkermansia muciniphila JCM 33894T strain was purchased from the Japan Microbial Collection (JCM).

[0050] See Table 1 for experimental instruments.

[0051] Table 1 Experimental Instruments

[0052] name model Origin and Company OLYMMPUS microscope BX53 Olympus Corporation, Japan centrifuge 5424R Germany, Eppendorf Constant temperature metal bath MK-20 China, Austria Micropipettes Germany, Eppendorf Mouse tail vein injection device KW-XXY China, Calvin refrigerator -20℃，-80℃ China, Haier Real-time PCR instrument CFXOPUS 96 United States, Bio-Rad Flow cytometer CytoFLEX Beckman Coulter, USA Ultra-high speed centrifuge Optima XE100 Beckman Coulter, USA

[0053] See Table 2 for experimental reagents and consumables.

[0054] Table 2 Experimental Reagents and Kits

[0055] name Manufacturer Giemsa dye China, Rain and Dew syringe China, Weigao cryopreservation tubes Thermo Fisher Scientific, USA glass slide China, Shitai methanol China, Zhongtian dimethyl sulfoxide United States, VWR Slide storage box China, Azure Sky PBS buffer powder UK, Servicebio pipette tips Thermo Fisher Scientific, USA fluorescently labeled antibodies United States, BioLegend Primers Azenta, USA Mirror oil China, Origin Heparin sodium micro-volume blood collection tube China, Kang Shifei

[0056] 2AKK bacterial culture and preparation of exovesicles

[0057] (1) AKK bacterial culture

[0058] The culture medium (3.85g brain heart extract, 1.6g soybean peptone, 1.13g anhydrous glucose, 0.55g N-acetylglucosamine, 0.4g L-threonine, 0.05g L-cysteine, 100mL) was used for anaerobic incubation at 37°C for 3 days until the bacterial suspension became slightly turbid. After counting, the bacterial pellet was collected by centrifugation, washed, and resuspended in PBS for later use.

[0059] (2) Preparation of AKK bacteria exovesicles

[0060] 400 mL of expanded AKK bacterial culture was centrifuged at 11000 g for 20 min, and the supernatant was collected. The supernatant was aliquoted into 50 mL centrifuge tubes, centrifuged at 11000 g for 10 min, and the supernatant was collected again. The supernatant was then centrifuged at 11000 g for 10 min using a vacuum filtration system through 0.45 μm and 0.22 μm filter membranes. The supernatant was then centrifuged at 200000 g for 130 min, and the supernatant was discarded. AKK-OMVs precipitate was visible. The AKK-OMVs were resuspended in PBS, and the concentration was determined using the BCA method. The precipitate was then stored at -80°C.

[0061] 3. Establishment of a mouse malaria model

[0062] (1) Resuscitation and passage: The PbA Plasmodium strain frozen in liquid nitrogen was placed in a 37°C water bath. After thawing, blood was drawn using a 1ml syringe and immediately inoculated intraperitoneally into C57BL / 6 mice at a dose of 0.2mL / mouse. This was the resuscitation of the strain. Blood smears were prepared from the terminal blood of the rat tails. The percentage of infected red blood cells in 1000 red blood cells was counted under different fields of view, which was the protozoemia density. When the protozoemia level reached 15%-30%, blood was collected from the eyeballs in heparin anticoagulant tubes and administered at 1×10 6 0.2 mL of iRBC-infected red blood cells per mouse were passaged into progeny C57BL / 6 mice.

[0063] (2) Establishment of a mouse model of PbA infection: When the protozoemia level in donor mice reaches 15%-30%, blood is collected from the eyeballs in heparin anticoagulant tubes, and then injected with 1×10 6 A mouse model of PbA infection was established by inoculating experimental mice with 0.2 ml of blood from one iRBC per mouse.

[0064] (3) Observation of protozoemia: Blood was collected from the tail vein and a thin blood smear was prepared. The blood smear was then stained with Giemsa stain. Protozoemia was assessed by examining the Giemsa-stained thin blood smear under an optical microscope with an eyepiece of 10x and an objective lens of 100x. Protozoemia was examined and quantified by counting the number of iRBCs in at least 1,000 red blood cells.

[0065] 4. Observation of macrophage phagocytosis of iRBCs in vitro

[0066] (1) Inducing macrophage polarization tendency

[0067] Use 2μL 10 9 CFU / mL AKK or 80μL 250μg / mL AKK exovesicles with 2×10 5 BV2 macrophages were co-cultured in 6-well plates at 37°C and 5% CO2 to induce polarization.

[0068] Subsequently, 10 μL of RBCs and iRBCs were added to the culture system.

[0069] Subsequently, fully lysed BV2 cells were collected using Trizol, and RT-qPCR experiments were performed using primers for six markers: TNF-α, TGF-β, CD86, CD206, iNOS, and Arg. The cell pellet after co-culture was collected, incubated with fluorescently labeled antibodies against F4 / 80, CD86, and CD206, and then analyzed by flow cytometry. Comparison with a control group containing only BV2 cells was conducted to determine the polarization trend of BV2 cells during co-culture to exert their immunomodulatory effects.

[0070] (2) Statistical analysis of macrophage phagocytosis rate of iRBCs

[0071] The phagocytic rate of iRBCs by BV2 cells was detected and calculated using flow cytometry. BV2 cells incubated with Far Red and treated iRBCs were mixed in 1.5 mL centrifuge tubes with serum-free medium and co-cultured at 37°C and 5% CO2 for 2 hours. After centrifugation, the supernatant was discarded, and the cells were washed twice with PBS to remove residual fluorescent dye. Each sample was processed according to flow cytometry procedures and then loaded onto the instrument. APC-positive cells were circled within the cell population (BV2 cells), and FITC-positive cells were circled within the BV2 cells (BV2 cells that had phagocytosed iRBCs). The software calculated the ratio of these two cell populations to determine the proportion of BV2 cells that had undergone phagocytosis.

[0072] 5. Observation of macrophage phagocytosis of iRBCs in vivo

[0073] Six- to eight-week-old C57BL / 6 mice were randomly divided into four groups of five each. After acclimatization, each mouse was intraperitoneally injected with 200 μL of passaged mouse erythrocytes at a worm density of 15%, and the day of injection was recorded as D0.

[0074] On the same day, each mouse in the BLK group was intraperitoneally injected with 200 μL of chlorphosphoside-free liposome PBS solution;

[0075] Each mouse in the NEG, M0, and M1 groups was injected intraperitoneally with 200 μL of chlorophosphonate liposomes to clear macrophages from the body.

[0076] Starting from D0, the mouse's weight needs to be recorded daily.

[0077] On day 3, each mouse in the BLK group was intraperitoneally injected with 200 μL of chlorphosphoside-free liposome PBS solution.

[0078] The NEG group continued to receive intraperitoneal injections of 200 μL clophosphate liposomes (same as D0), while each mouse in the M0 group received an intraperitoneal injection of 10 μL clophosphate liposomes. 6200 μL of BV2 cells in the M0 state were injected intraperitoneally into each mouse in the M1 group. 6 BV2 cells in M1 polarization state after treatment with 200 μL LAKK-OMVs.

[0079] Starting from day 3, blood smears were prepared daily to count protozoemia in each group, and body weight and RMCBS scores were measured regularly until the mice began to die on day 6.

[0080] 5 Experimental Results

[0081] 5.1 Successful culture of AKK bacteria and extraction of AKK-OMVs

[0082] After three passages and expansion culture, AKK-OMVs were obtained at a concentration of 8 mg / mL by filtration and ultracentrifugation using the BCA method. Figure 2 Meanwhile, the logarithmic-phase AKK bacteria were cryopreserved for subsequent experiments.

[0083] 5.2 A mouse model of infection with Plasmodium was successfully established.

[0084] Resuscitate the ANKA strain of Plasmodium berghei (PbA) from liquid nitrogen cryopreservation and infect 6-8 week old female C57BL / 6 mice. Protozoemia is assessed by blood smear analysis. Mice are passaged when protozoemia is >15%. Mice from the third passage are used as experimental subjects. A model is considered successfully established when protozoemia is >15% and accompanied by typical neurological symptoms. Figure 2 .

[0085] 5.3 RT-qPCR verification of the polarization and depolarization effects of AKK and its outer vesicles on macrophages

[0086] 5.3.1 RT-qPCR verification of macrophage polarization

[0087] In the co-culture experiment, statistical analysis of the relative expression levels between groups showed that the experimental groups with added AKK bacteria and AKK-OMVs exhibited a trend of macrophage M1 polarization, and AKK-OMVs had a stronger ability to induce macrophage M1 polarization than AKK bacteria. Figure 3 As shown, the target genes indicating M1 are iNOS, CD86, and TNF-α, while the target genes indicating M2 are Arg, CD206, and TGF-β. AKK bacteria and AKK-OMVs can inhibit the expression of M2-related genes, and the expression levels of M2-related genes are even lower in the AKK-OMVs group.

[0088] 5.3.2 RT-qPCR verification of macrophage reverse polarization

[0089] After confirming that AKK bacteria or AKK-OMVs can polarize macrophages to M1, a reversal experiment was designed to verify whether macrophages can transform from M2 to M1 type under the influence of AKK bacteria or AKK-OMVs. After providing an M2-polarized culture environment for macrophages, AKK and AKK-OMVs were added. Based on the relative expression levels of different target genes, AKK and AKK-OMVs still had varying degrees of M1 polarization conversion effects on macrophages that had already completed M2 polarization, with AKK-OMVs showing a better reversal effect. Mass spectrometry analysis of AKK-OMVs revealed the presence of multiple active components, including Amuc_1100, which may affect macrophage function and activity. Figure 4 As shown.

[0090] 5.4 Flow cytometry verification of the polarization and depolarization effects of AKK and AKK-OMVs on macrophages

[0091] 5.4.1 Flow cytometry verification of macrophage polarization by AKK and AKK-OMVs

[0092] After co-culturing as described above, BV2 cells were collected. CD86 and CD206 were used as fluorescent dyes. After incubation, the cells were analyzed by flow cytometry. The results showed that BV2 cells in both the AKK and AKK-OMVs groups exhibited M1 polarization, consistent with RT-qPCR results. Figure 5 As shown.

[0093] 5.4.2 Flow cytometry verification of the depolarization effect of AKK and AKK-OMVs on M2 macrophages

[0094] After co-culturing as described above, BV2 cells were collected. CD86 and CD206 were used as fluorescent dyes. After incubation, the cells were analyzed by flow cytometry. The results showed that AKK and AKK-OMVs tended to reverse M2 polarization to M1 polarization in each group, with AKK-OMVs showing a more pronounced effect, consistent with RT-qPCR results. Figure 6 As shown.

[0095] 5.5 Observation of macrophage phagocytosis in vitro and statistical analysis of phagocytosis rate

[0096] 5.5.1 Morphological observation under an optical microscope

[0097] After co-culturing as described above, BV2 cells were collected by centrifugation. Six smears containing both thick and thin blood membranes were prepared from each group. After drying, the smears were fixed with formaldehyde for 20 seconds. Freshly prepared Giemsa stain (Giemsa:PBS = 1:10) was evenly applied to the smears, and staining was performed for 10 minutes. The smears were then rinsed slowly with a fine stream of water, avoiding direct contact between the water and the smears. After rinsing for 1 minute, the smears were allowed to dry. The phagocytic activity of macrophages on each smear was observed as follows. Figure 7Fifty fields of view were randomly photographed from each smear for phagocytosis rate analysis. The phagocytosis rates of the AKK group and the AKK-OMVs group were significantly higher than those of the CTL group. Compared to the AKK group, the AKK-OMVs group showed no significant difference in phagocytosis of iRBCs, suggesting that AKK-OMVs can be used to replace AKK bacteria in inducing M1 polarization in macrophages, promoting the latter's phagocytosis of iRBCs. Unstained phagocytic smears from co-culture were prepared, such as... Figure 7 As shown.

[0098] 5.5.2 Morphological observation of phagocytosis under scanning electron microscopy (see [reference needed]). Figure 8 -A, B, C. This figure shows optical microscopy, scanning electron microscopy, and transmission electron microscopy images of BV2 cells phagocytizing iRBCs after AKK treatment.

[0099] 5.5.3 The morphological observation under transmission electron microscopy is shown in the figure. Figure 8 -D, E, F. This figure shows optical microscopy, scanning electron microscopy, and transmission electron microscopy images of BV2 cells phagocytizing iRBCs after treatment with AKK-OMVs.

[0100] 5.6 Observation of phagocytosis of infected erythrocytes by polarized macrophages in vivo

[0101] As the number of days after infection increased, all experimental groups except the BLK group experienced varying degrees of weight loss on day 1 after injection of macrophage scavengers, but most groups regained weight on day 2. After day 3, the weight of all C57BL / 6 mice showed a decreasing trend, with the M1 group showing a relatively stable decrease, while the BLK group experienced a larger decrease in the later stages. The RMCBS score began to decrease on day 3, and the score decreased significantly after day 4. The score of the M1 group did not show a significant decrease until day 5. Protozoemia was recorded from day 3. Compared with the BLK group, the groups injected with macrophage scavengers had slightly higher levels of protozoemia. The increase in protozoemia in the M1 group was relatively slower. The BLK group died on day 6, while the NEG, M0, and M1 groups died on day 7. Figure 9 .

[0102] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of *Ackermania pseudomalaris* exovesicles in the preparation of antimalarial drugs, characterized in that, The Akkermansia muciniphila strain is Akkermansia muciniphila JCM 33894T.

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