Application of Lactobacillus murinus-driven GABA in the treatment of cerebral malaria in mice

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to the application of Lactobacillus murinus driving GABA in the treatment of cerebral malaria in mice. The present invention found significantly enriched Lactobacillus murinus and confirmed that Lactobacillus murinus can produce GABA. Subsequently, in a mouse cerebral malaria model, the effects of Lactobacillus murinus and GABA on improving and treating mouse cerebral malaria were evaluated when Lactobacillus murinus and GABA were used alone or in conjunction with dihydroartemisinin. The results showed that whether used alone or in combination with dihydroartemisinin, the bacteria and its metabolite GABA 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. From the perspective of using probiotics to drive their metabolites to assist antimalarial drugs in the treatment of mouse cerebral malaria, the present invention provides a new solution for the treatment of severe malaria caused by falciparum malaria, especially human cerebral malaria.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to an application of Lactobacillus murinus driving GABA in treating cerebral malaria in mice. Background Art

[0002] Cerebral malaria (CM) is a neurological term introduced in 2020. It is a parasitic brain disease caused by direct invasion of malarial parasites, particularly Plasmodium falciparum. It is a severe disease with a high mortality rate, making it one of the most severe forms of malaria. Approximately 1% to 2% of cases of falciparum malaria will eventually develop into cerebral malaria, which occurs primarily in children under 5 years of age in Africa and has a mortality rate of approximately 15% to 20%. Clinical manifestations of cerebral malaria may include hemiparesis, convulsions, ataxia, coma, impaired consciousness, meningeal irritation, and even death. Even with early treatment with standard antimalarial chemotherapy, the mortality rate remains very high after the parasite is cleared from the body. Furthermore, approximately 25% of survivors experience neurological complications and cognitive impairment. Among surviving children, 10% to 20% experience persistent neurological deficits, cognitive impairment, behavioral disturbances, and motor impairment.

[0003] Currently, quinine and artemisinin are recommended treatments for CM. Although artemisinin-based drugs have highly effective antimalarial effects, the parasites that die after killing them remain trapped in local microvasculature. The resulting ischemic changes in the brain and microvascular damage cannot be effectively repaired, resulting in mortality rates as high as 18% and 30% in children and adults with cerebral malaria, respectively. Studies have also shown that artemisinin-based combination therapies (ACTs) often fail to protect against cell death, neurological damage, and cognitive deficits. Therefore, artemisinin-based drugs alone are insufficient to prevent death or neurological impairment in all patients with cerebral malaria. This suggests that, building on existing treatment strategies, we need to continue exploring novel artemisinin-based drug combinations, treatment regimens, and adjunctive therapies.

[0004] In the early stage, we conducted a series of exploratory studies on mouse malaria, especially the mouse cerebral malaria model caused by Plasmodium berghei. Studies have shown that falciparum malaria can cause intestinal damage and is related to intestinal flora and its metabolites, but the mechanism is unclear. Subsequently, the applicant observed and analyzed the changes in the species and abundance of intestinal flora in different mouse malaria models. The results showed that after infection with Plasmodium, the species of intestinal flora in mice decreased, the abundance of probiotics decreased, and harmful bacteria increased. Subsequently, based on the previous research, we continued to explore the effects and functions of different drug combinations of dihydroartemisinin (DHA), rapamycin (RAP) and atorvastatin (AVA) on the intestinal flora in the mouse cerebral malaria model. The results showed that Lactobacillus murinus was significantly enriched after the use of dihydroartemisinin. However, it is not clear whether this bacterium plays a role in the treatment of mouse cerebral malaria and how it plays a role. To this end, the present invention intends to establish a mouse cerebral malaria model, use the bacteria for intervention, and evaluate the effect of Lactobacillus murinus in treating mouse cerebral malaria from the perspectives of behavior and pathology, in order to provide an effective solution for the treatment of human cerebral malaria.

[0005] Studies have shown that intestinal probiotics play an important role in intestinal inflammatory diseases, tumors, and neurological diseases. However, there are no reports on the role of intestinal probiotics, especially whether and how Lactobacillus murinus plays a role in infectious diseases, especially cerebral malaria. The reason for the lack of research may be that the use of intestinal probiotics to treat malaria is an interdisciplinary subject. Experts and scholars in the field of malaria are more concerned about the molecular biological basis of Plasmodium pathogenicity. In terms of treatment, more attention is focused on developing new drugs to replace artemisinin and its derivatives, or on studying vaccines for the prevention or treatment of malaria through various technical means. At present, there have been some progress in the above, but there is still a certain distance from actual clinical treatment. The present invention is inspired by the treatment of major diseases including tumors by intestinal flora, and has carried out research on the interaction between intestinal flora and parasitic infections in China earlier.

[0006] To this end, the present invention seeks to reveal the role and possible mechanism of action of Lactobacillus murinus in the treatment of cerebral malaria in mice. Specifically, a mouse cerebral malaria model was first established. This model was then treated with either Lactobacillus murinus, GABA, combined with dihydroartemisinin, or with either Lactobacillus murinus or GABA alone. The therapeutic efficacy of Lactobacillus murinus against cerebral malaria was observed using behavioral, pathological, and molecular biological techniques. Summary of the Invention

[0007] The present invention first provides an application of Lactobacillus murinus driving GABA in treating cerebral malaria in mice, wherein the application is in vivo or in vitro;

[0008] Optionally, the treatment of mouse cerebral malaria comprises one or more of the following:

[0009] A) Improve mouse behavior;

[0010] B) Improve immune pathological damage to various organs;

[0011] C) reduce parasitemia;

[0012] D) Prolong the survival of mice.

[0013] In certain embodiments, the in vitro is bacteria, and the in vivo is in mice.

[0014] The present invention also provides the use of GABA in treating cerebral malaria in mice, wherein the use is in vivo;

[0015] Optionally, the treatment of mouse cerebral malaria comprises one or more of the following:

[0016] A) Improve mouse behavior;

[0017] B) Improve immune pathological damage to various organs;

[0018] C) reduce parasitemia;

[0019] D) Prolong the survival of mice.

[0020] Optionally, the GABA is prepared from Lactobacillus murinus or purchased from a company.

[0021] The present invention also provides the use of Lactobacillus murinus or GABA in the preparation of a medicine for improving and treating cerebral malaria caused by Plasmodium berghei in treating cerebral malaria in mice.

[0022] In certain embodiments, the mouse cerebral malaria is cerebral malaria caused by Plasmodium berghei.

[0023] In certain embodiments, the drug further comprises one or three of dihydroartemisinin, rapamycin, and atorvastatin.

[0024] In certain embodiments, the drug must be dihydroartemisinin.

[0025] The present invention also provides a pharmaceutical composition for treating mouse cerebral malaria, wherein the medicine comprises Lactobacillus murinus and / or GABA.

[0026] In certain embodiments, the pharmaceutical composition further comprises dihydroartemisinin, rapamycin, and atorvastatin.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] The present invention, through combined analysis of intestinal flora and metabolomics, identified significantly enriched Lactobacillus murinus and its metabolite GABA. Subsequently, we confirmed that Lactobacillus murinus can produce GABA. Subsequently, we evaluated the efficacy of Lactobacillus murinus and GABA in improving and treating cerebral malaria in a mouse cerebral malaria model, using them alone or in combination with dihydroartemisinin. The results suggest that the bacteria and its metabolite GABA, whether used alone or in combination with dihydroartemisinin, can effectively improve mouse behavior, immune pathological damage to various organs, reduce parasitemia, and prolong mouse survival, effectively treating cerebral malaria in mice compared to using either drug alone. Since the Lactobacillus murinus used is a common probiotic lactobacillus and dihydroartemisinin is a first-line clinical antimalarial drug, clinical translation for the treatment of human cerebral malaria can shorten the conversion time. This invention is based on the concept that metabolites produced by probiotics can assist in the treatment of malaria with drugs, and from this perspective, provides a solution for the treatment of human cerebral malaria. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Experimental groups and treatment plan. C57BL / 6 mice were infected with PbA to establish an ECM model, with the day of infection being designated as day 0. Drug treatment was administered on day 3 post-infection, and L. Murinus and GABA were administered orally on day 0 post-infection. Days 3 to 7 post-infection were the ECM stage of synergistic bacterial and drug treatment. The drugs included dihydroartemisinin, rapamycin, and atorvastatin. The rectangular boxes in the figure represent the collection of feces from each group of mice on day 9 post-infection and on day 10 for histopathological observation and assessment of intestinal barrier integrity.

[0030] Figure 2 Stacked bar chart of relative species abundance at the genus level

[0031] Figure 3 Analysis of changes in brain metabolites. DHA, DHA, RAP, and AVA represent levels after infection with Plasmodium berghei and drug treatment, respectively. The red arrow points to GABA.

[0032] Figure 4 Correlation analysis between bacterial flora and metabolites. From left to right: PbA group, DHA group, and DHA.RAP.AVA group. GABA was positively correlated with Lactobacillus, R² = 0.8375, P = 0.2642; GABA was positively correlated with Lactobacillus murinus, R² = 1.000, P = 0.0018.

[0033] Figure 5 Gram staining of Lactobacillus murine culture in vitro (1000×)

[0034] Figure 6Monitoring of relevant indicators in the ECM model treated with different bacterial-drug combinations. (A) Body weight changes in ECM mice treated with different bacterial-drug combinations; (B) RMCBS score curves for ECM mice treated with different bacterial-drug combinations; (C) Protozoalemia in ECM mice treated with different bacterial-drug combinations; (D) Survival analysis of ECM mice treated with different bacterial-drug combinations. Survival was analyzed using the log-rank (Mantel-Cox) test. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0035] Figure 7 Semiquantitative analysis of hemozoin and red pulp area. (A) Analysis of the hemozoin ratio in the liver of the different bacterial and drug treatment groups; (B) Analysis of the hemozoin ratio in the spleen of the different treatment groups; (C) Quantitative analysis of the red pulp (RePu) area of ​​the different treatment groups. Statistical analysis was performed using one-way ANOVA, with the LSD post hoc analysis used for each analysis. Error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0036] Figure 8 Relevant intestinal parameters of the ECM model treated with different bacterial drugs. (A) Intestinal villus length of ECM mice receiving different treatments; (B) Crypt depth of the colon of ECM mice receiving different treatments; (C) Goblet cells of the colon of ECM mice receiving different treatments; (D) Intestinal permeability analysis of ECM mice. Statistical analysis was performed using one-way ANOVA with a LSD post hoc test for each one-way ANOVA. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0037] Figure 9 HE staining of intestinal tissue (100×). The orange rectangle indicates the destruction of intestinal villi and the absence of goblet cells. The red arrow indicates the separation of intestinal epithelial cells. Scale bar: 50 μm.

[0038] Figure 10Histopathological observations of the mouse brain, liver, and spleen. (A) Hematoxylin and eosin (HE) staining of the brain (magnification, ×1,000). Purple arrows indicate red blood cells (RBCs), black arrows indicate brain microvascular endothelial cells (BMECs), green oval boxes represent rosetting of iRBCs and leukocytes, red rectangular boxes represent leukocyte obstruction, and yellow oval boxes represent hemorrhage. Scale bar: 10 μm. (B) HE staining of the liver (magnification, ×400). Red arrows represent normal red blood cells in venous vessels, black arrows represent iRBCs, yellow rectangular boxes represent hemoglobin, red rectangular boxes represent fat vacuoles, and blue oval circles represent adhesion and aggregation of leukocytes and iRBCs. Scale bar: 20 μm. (C) HE staining of the spleen (magnification, ×400). Red areas represent RePu, and blue areas represent white pulp (WhPu). Scale bar: 20 μm. DETAILED DESCRIPTION

[0039] 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.

[0040] Abbreviations:

[0041] DHA: dihydroartemisinin;

[0042] RAP: rapamycin;

[0043] AVA: atorvastatin;

[0044] Example 1. Materials and methods

[0045] 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.

[0046] The Plasmodium berghei ANKA strain 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.

[0047] Lactobacillus murinus (Cat. No. BNCC194688) was purchased from Beijing Anxinkang Technology Co., Ltd.

[0048] GABA (catalog number: DC12615-5g) was purchased from Shanghai Xushuo Biotechnology Co., Ltd.

[0049] The experimental instruments are shown in Table 1.

[0050] Table 1 Experimental instruments

[0051] name model Origin and company scales 76175 China, Beardsman OLYMMPUS microscope BX53 Olympus Corporation, Japan centrifuge 5424R Eppendorf, Germany Constant temperature metal bath MK-20 China, Aosheng Tissue Slice Imaging System BX53 Olympus Corporation, Japan Micropipette 2-volume pipette US imaging system Eppendorf, Germany Multifunctional microplate reader SYNERGY-HT BioTek, USA Mouse tail vein injection instrument KW-XXY China, Calvin refrigerator -20℃，-80℃ Haier, China

[0052] The experimental reagents and consumables are shown in Table 2.

[0053] Table 2 Experimental reagents and kits

[0054] 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

[0055] Example 2

[0056] (1) Recovery 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 recovery. When the parasitemia level of the blood seed mice reaches 15%-30%, remove the eyeballs and draw blood into heparin anticoagulant tubes. 6 The iRBCs-infected erythrocytes were passaged into offspring C57BL / 6 mice at a dose of 0.2 ml per mouse.

[0057] (2) Exploration of intestinal flora and metabolites

[0058] (2.1) Experimental grouping and drug treatment: This grouping was mainly used for intestinal flora and metabolite exploration, as follows: 30 female C57BL / 6 mice were randomly divided into three groups: infection-untreated group (PbA), DHA-treated group (DHA), and DHA combined with RAP and AVA-treated group (DHA.RAP.AVA).

[0059] DHA, RAP, and AVA were dissolved in 5% DMSO and 0.9% NaCl at 3 mg / kg, 5 mg / kg, and 40 mg / kg, respectively. On day 3 post-infection, the PbA group received an intraperitoneal injection of 5% DMSO as a control. The DHA group received 3 mg / kg DHA. The DHA, RAP, and AVA group received 5 mg / kg RAP, 3 mg / kg DHA, and 40 mg / kg AVA. These mice were treated with these drugs intraperitoneally for 5 consecutive days.

[0060] (2.2) Sample Collection and Sequencing Analysis: On day 8 post-infection, fecal pellets from each group of mice were collected using sterile forceps, placed in sterile centrifuge tubes, and immediately frozen in liquid nitrogen and stored at -80°C until use. Concurrently, mouse brain tissue was collected for metabolomics sequencing. Subsequently, the gut microbiome sequencing results were combined with brain metabolite analysis.

[0061] (3) Observation on the treatment of cerebral malaria in mice with rat lactate and its metabolite GABA

[0062] (3.1) Lactobacillus murinus culture

[0063] Lactobacillus murinus (Cat. No. BNCC194688) was cultured in MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and incubated anaerobically at 37°C for 24 hours. The MRS-cultured bacteria (5% v / v) were then inoculated into CM medium and incubated anaerobically at 37°C for 48 hours. After incubation, the bacteria were harvested by centrifugation, resuspended in a suspension solution (50 g / L skim milk, 30 g / L lactose, 50 g / L yeast extract, and 50 g / L ascorbic acid), and freeze-dried for storage at -80°C. Meanwhile, 100 μL of the bacterial suspension was centrifuged at 3000 g for 5 minutes, resuspended in 20 μL PBS, and 10 μL was stained with Gram stain for microscopic examination.

[0064] (3.2) Detection of GABA, a metabolite of Lactobacillus murinus

[0065] Culture L. murinus as before. Take a certain amount of bacterial culture and detect the GABA content in the bacteria according to the instructions of the "Bacterial Gamma-Aminobutyric Acid (GABA) Content Fluorescence Quantitative Detection Kit".

[0066] (3.3) Experimental groups and drug treatment

[0067] This grouping is mainly used to evaluate the effects of bacteria and their metabolites in treating cerebral malaria in mice. Specifically, according to different treatment strategies, mice were randomly divided into 6 groups: PbA group, PbA+Lmu group, PbA+GABA group, DHA group, DHA+Lmu group, and DHA+GABA group.

[0068] After infection of C57BL6 / N mice, mice in the PbA group were given 2.5% glycerol and 5% dimethyl sulfoxide on day 0 and day 3 after infection, respectively. Mice in the PbA+Lmu group were given 6.8×10 8 CFU / mL of L. Murinus and 5% DMSO. The PbA+GABA group was given 30 mg / kg GABA and 5% DMSO on days 0 and 3 after infection, respectively. The DHA group was given 2.5% glycerol and 3 mg / kg DHA on days 0 and 3 after infection, respectively. The DHA+Lmu group was given 6.8×10 8CFU / mL of L. Murinus and 3mg / kg DHA. The DHA+GABA group was treated with 30mg / kg GABA and 3mg / kg DHA, respectively. Each drug was administered for 5 consecutive days, with a single dose of 200μL. L. Murinus and GABA were administered by gavage starting from day 0 until the mice died, with a single gavage dose of 200μL. Phenotypic and behavioral records were performed every day, and the results are shown in Figure 1 .

[0069] (4) Basic indicator testing

[0070] Prostozoaemia: Blood was collected from the tail vein and thin blood smears were prepared and stained with Giemsa. Prostozoaemia was assessed under a light microscope at 100x magnification. Prostozoaemia was examined and quantified by counting the number of iRBCs in at least 1,000 red blood cells (RBCs).

[0071] Basic behavioral assessment: Starting on day 0 post-infection, mice were monitored daily for body weight, neurological characteristics, parasitemia, and survival. Neurological characteristics were assessed using the Rapid Murine Coma and Behavioral Scale (RMCBS), which utilizes 10 parameters (hair, limb strength, defensive ability, auricular reflex, toe reflex, touch reflex, gait, balance, body posture, and exploratory activity). ECM was successfully established if mice gradually developed unsteady gait, ataxia, matted hair, inability to stretch, absent toe reflex, absent auricular reflex, convulsions, coma, or even death. Parasitemia was assessed and quantified by counting the number of iRBCs in 1,000 red blood cells (RBCs) in a thin blood film smear under a 100× optical microscope.

[0072] Blood-brain barrier integrity assessment: The protective effect on the brain was assessed by blood-brain barrier integrity (BBB). 1% EB dye was diluted in 0.9% NaCl. On day 8 after infection, 200 μl of 1% EB dye solution was injected into the tail vein of mice. The dye was allowed to circulate for 30 minutes. The mice were then anesthetized and perfused into the right atrium through the heart with 0.9% NaCl to allow the liquid to flow out. The brains were quickly isolated, 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 incubated in a thermostatic 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.

[0073] Histopathological Observations: Tissue samples were collected after administration, i.e., on day 8 post-infection. Immediately after euthanasia, brain, liver, spleen, and small intestinal tissues were collected and washed three times in cold PBS to remove blood. The tissues were then fixed with a universal tissue fixative for 24 hours and embedded in paraffin. Serial 4-μm-thick sections were cut and stained with hematoxylin and eosin to observe microvascular obstruction and leakage. Sections were observed using a light microscope, and images were acquired using Olympus cellSens standard 1.13 software.

[0074] result:

[0075] (1) Changes in intestinal flora

[0076] After the intervention experiment with dihydroartemisinin, rapamycin, and atorvastatin, the feces of mice were collected for 16S RNA sequencing. Intestinal flora analysis found that the abundance of Lactobacillus gradually increased in the infection-untreated group, the DHA single-drug group, and the three-drug combination group (DHA, RAP, AVA), indicating that L. murinus was still a significantly enriched species. The results are shown in Figure 2 .

[0077] (2) Changes in metabolites

[0078] At the same time, mouse brain tissue was collected for metabolomics sequencing. Figure 3 As shown in the figure, brain metabolome analysis revealed that GABA levels also showed an increasing trend in the untreated infection group, the DHA monotherapy group, and the DHA, RAP, and AVA groups (indicated by the red arrows). This suggests that this metabolite plays a positive role in the treatment of cerebral malaria caused by Plasmodium berghei infection.

[0079] (3) Combined analysis of intestinal flora and metabolomics

[0080] See also Figure 4 Linear regression analysis showed that GABA and Lactobacillus murinus were positively correlated in the infection-untreated group, DHA monotherapy group, and DHA.RAP.AVA group (R 2 =1.00, P=0.0018). 2 =0.8375, P=0.2462), especially Lactobacillus johnsonii (R 2 Whether there is an underlying mechanism behind this positive correlation requires further investigation.

[0081] Example 3 Observation on the Treatment of Cerebral Malaria in Mice by Lactate and Its Metabolite GABA

[0082] (1) Microscopic morphology of Lactobacillus murinus Figure 5 shown.

[0083] (2) Detection of GABA, a metabolite of Lactobacillus murinus

[0084] Testing with the kit revealed GABA levels of 2.595 mmol / mL and 2.251 mmol / mL at 0 and 10 minutes, respectively. This is consistent with GABA levels reported in the literature for other lactobacilli. These results provide preliminary evidence that Lactobacillus murinus can produce GABA.

[0085] (3) Effects of Lactobacillus murinus and its metabolite GABA on ECM

[0086] exist Figure 6 In the DHA+Lmu group (using both DHA and Lmu in a mouse cerebral malaria model), the average body weight of mice increased by 25.35% [(DHA+Lmu group - PbA+Lmu group) / PbA+Lmu group] and 5.25% [(DHA+Lmu group - DHA group) / DHA group] on day 9, compared with the PbA+Lmu group (using only Lmu in a mouse cerebral malaria model) and the DHA group (using only DHA in a mouse cerebral malaria model). On day 10, the RMCBS scores of mice increased by 282.54% [(DHA+Lmu group - PbA+Lmu group) / PbA+Lmu group] and 1.36% [(DHA+Lmu group - DHA group) / DHA group], respectively. On the 8th day, the parasitemia of mice was reduced by 85.85% [(DHA+Lmu group-PbA+Lmu group) / PbA+Lmu group] and 15.24% [(DHA+Lmu group-DHA group) / DHA group], respectively.

[0087] Compared with the PbA+GABA group (GABA intervention alone) and the DHA group (DHA treatment alone), the average body weight of mice in the DHA+GABA group (DHA+GABA group - PbA+GABA group) and DHA group (DHA treatment alone) increased by 28.6% [(DHA+GABA group - PbA+GABA group) / PbA+GABA group] and 3.16% [(DHA+GABA group - DHA group) / DHA group] on day 9, respectively. On day 7, the RMCBS scores of mice increased by 205.17% [(DHA+GABA group - PbA+GABA group) / PbA+GABA group] and 5.92% [(DHA+GABA group - DHA group) / DHA group], respectively. On the 8th day, the parasitemia of the mice was reduced by 86.75% [(DHA+GAB group-PbA+GABA group) / PbA+GABA group] and 28.03% [(DHA+GABA group-DHA group) / DHA group], respectively. The results are shown in Table 3.

[0088] Table 3. Basic indicator average value record table

[0089] Group Weight(D9) RMCBS score (D10) Protozoaemia (D8) PbA 13.644 1.800 51.74 PbA+Lmu 16.100 2.933 44.04 PbA+GABA 15.430 -- 39.93 DHA 19.176 11.070 7.35 DHA+Lmu 20.182 11.22 6.23 DHA+GABA 19.782 11.78 5.29

[0090] Note: The time selected in the table is based on the data of the PbA+Lmu group, where D8, D9 and D10 represent the days after infection.

[0091] like Figure 6 As shown in Figure A, starting from day 0 post-infection, the body weight of mice in all drug-treated groups showed an overall trend of first decreasing, then increasing, and then decreasing again compared to the untreated group. The average body weight of the PbA+Lmu group was higher than that of the PbA+GABA group, but the difference was not statistically significant. The average body weight of the DHA+Lmu group was higher than that of the DHA group, with a significant difference between the two groups (P=0.0006). The average body weight of the DHA+GABA group was higher than that of the DHA group, but the difference was not statistically significant (P=0.389). The average body weight of the DHA+Lmu group was higher than that of the DHA+GABA group, but the difference was not statistically significant (P=0.555). Among the combined bacterial and drug treatment groups, the DHA+Lmu group had a slight decrease in body weight, but the decrease was the smallest, while the DHA group had the largest decrease in body weight, with a statistically significant difference between the two groups (P=0.031).

[0092] The Rapid Mouse Coma and Behavior Scale (RMCBS) was used to assess the manifestation of ECM. Figure 6 As shown in Figure B, the RMCBS scores of mice in all groups showed a downward trend. Mice in the PbA group began to show symptoms of decreased exploratory ability, weakened limb strength, and unsteady gait on day 4 post-infection. Their RMCBS scores rapidly declined with the onset of CM symptoms. On day 7 post-infection, the RMCBS scores of mice in the PbA group were below 4, consistent with the symptoms exhibited by the mice: decreased exploratory behavior, diminished reflexes, decreased self-defense ability, coma, and convulsions. On day 10 post-infection, the RMCBS scores of mice were only 1.8, while those in the PbA+Lmu and PbA+GABA groups were significantly higher during the same period. RMCBS scores of mice in the drug-treated groups decreased slightly from day 4 to day 7 post-infection but remained within the normal range, with no statistically significant differences among the three groups. Later in the infection period, scores in the DHA group decreased more rapidly than those in the DHA+Lmu and DHA+GABA groups. On day 16 post-infection, the RMCBS scores in the DHA+Lmu group were significantly higher than those in the DHA group (P = 0.003). There was no statistically significant difference in RMCBS scores between the DHA+GABA group and the DHA group (P=0.106). Although the RMCBS score of the DHA+Lmu group was better than that of the DHA+GABA group, there was no significant difference between the two groups (P=0.083).

[0093] Figure 6Peripheral blood parasitemia results (Figure C) show an increasing trend in all groups, reaching an initial peak within the first three days of infection. Parasitemia in the PbA group began to rise rapidly from day 2 post-infection, peaking on day 8 post-infection, demonstrating a typical parasite growth pattern. In contrast, parasitemia in drug-treated mice decreased between the initial peak and the later proliferation phase. On day 8 post-infection, parasitemia in the PbA+Lmu group (P=0.068) and the PbA+GABA group (P=0.050) was significantly lower than that in the PbA group. Parasitemia in the DHA+GABA group was significantly lower than that in the DHA group (P=0.024). Parasitemia in the DHA+Lmu group was slightly lower than that in the DHA group, with no significant difference between the two groups (P=0.181). There was no significant difference between the DHA+GABA group and the DHA+Lmu group (P=0.217).

[0094] like Figure 6 As shown in Figure 2, mice infected with PbA developed neurological symptoms on day 4 post-infection (PI), leading to mortality between days 5 and 10. Approximately 50% of mice succumbed to ECM within 8 days post-infection, with parasitemia rates reaching 48.96-56.73%. All mice in the PbA group developed typical symptoms of ECM and died within 10 days post-infection. The survival rate of the PbA+Lmu group was significantly higher than that of the PbA group (P = 0.016). The survival time of the PbA+Lmu group was longer than that of the PbA+GABA group (P = 0.005). The mean survival time of the PbA group (8.10 ± 2.12) was higher than that of the PbA+GABA group (7.20 ± 1.64), but the difference was not statistically significant (P = 0.397). In the DHA combined with L. murinus and GABA regimen, all mice survived for 18 days. There was no statistically significant difference in survival rate between the DHA group and the DHA+Lmu group (P=0.607), the DHA+GABA group and the DHA group (P=0.213), and the DHA+Lmu group and the DHA+GABA group (P=0.223).

[0095] exist Figure 7In the DHA+Lmu group (a mouse model of cerebral malaria treated with both DHA and Lmu), compared with the PbA+Lmu group (a mouse model of cerebral malaria treated with only Lmu) and the DHA group (a mouse model of cerebral malaria treated with only DHA), the hemochromatin content in the liver of the mice decreased by 54.9% [(DHA+Lmu group - PbA+Lmu group) / PbA+Lmu group] and 22.14% [(DHA+Lmu group - DHA group) / DHA group], respectively. The hemochromatin content in the spleen of the mice decreased by 60.15% [(DHA+Lmu group - PbA+Lmu group) / PbA+Lmu group] and 29.92% [(DHA+Lmu group - DHA group) / DHA group], respectively. The red pulp area of ​​the spleen of mice increased by 31.95% [(DHA+Lmu group-PbA+Lmu group) / PbA+Lmu group] and 19.22% [(DHA+Lmu group-DHA group) / DHA group], respectively.

[0096] Compared with the DHA+GABA group (DHA and GABA intervention in the mouse cerebral malaria model), the PbA+GABA group (GABA intervention in the mouse cerebral malaria model) and the DHA group (DHA treatment in the mouse cerebral malaria model) showed that the hemochromatin content in the liver of the mice decreased by 54.88% [(DHA+GABA group - PbA+GABA group) / PbA+GABA group] and 29.27% ​​[(DHA+GABA group - DHA group) / DHA group], respectively. The hemochromatin content in the spleen of the mice decreased by 49.42% [(DHA+GABA group - PbA+GABA group) / PbA+GABA group] and 26% [(DHA+GABA group - DHA group) / DHA group], respectively. The red pulp area of ​​the spleen of mice increased by 33.65% [(DHA+GABA group-PbA+GABA group) / PbA+GABA group] and 25.17% [(DHA+GABA group-DHA group) / DHA group], respectively.

[0097] Figure 7 A is a quantitative analysis of the proportion of hemozoins in hepatic tissue pathological sections stained with hematoxylin and eosin. PbA combined with L. murinus was significantly more effective in clearing hepatic hemozoins than the PbA group, with a significant difference between the two groups (P < 0.0001). There was no significant difference in the proportion of hemozoins in the PbA + Lmu group and the PbA + GABA group (P = 0.244). DHA combined with L. murinus was significantly more effective in clearing hemozoins than the DHA group, with a significant difference between the two groups (P = 0.0011). DHA combined with GABA was significantly more effective in metabolizing hemozoins than the DHA group, with a significant difference between the two groups (P = 0.0003). There was no statistically significant difference in hemozoins between the DHA + Lmu group and the DHA + GABA group (P = 0.306).

[0098] Figure 7 B is a quantitative analysis of the proportion of hemozoin in spleen tissue. The PbA + Lmu group (P < 0.0001) and the PbA + GABA group (P < 0.0001) were both more capable of removing hemozoin than the PbA group. The hemozoin proportion in the PbA + Lmu group was higher than that in the PbA + GABA group, but the difference between the two groups was not statistically significant (P = 0.098). Compared with the DHA group, the hemozoin proportion in the DHA + Lmu group (P = 0.003) and the DHA + GABA group (P = 0.006) was lower. The difference between the DHA + Lmu group and the DHA + GABA group was not statistically significant (P = 0.26).

[0099] like Figure 7 As shown in Figure C, the RePu area in the spleen was quantitatively analyzed. There were significant differences in the RePu area between the PbA group and the PbA+Lmu group (P=0.0002) and the PbA+GABA group (P=0.0007). The RePu area in the PbA+GABA group was smaller than that in the PbA+Lmu group, but there was no significant difference between the two groups (P=0.222). Compared with the DHA group, the RePu area was significantly reduced in the DHA+Lmu group (P=0.0002) and the DHA+GABA group (P=0.0002). There was also a significant difference between the DHA+Lmu group and the DHA+GABA group (P=0.016). These results indicate that the DHA+Lmu group and the DHA+GABA group have more effective protective effects on the spleen compared with the DHA group.

[0100] like Figure 8 As shown, the length of the villi stained by HE was quantitatively analyzed. Compared with the control group, the villi length of all ECM mice was shortened. There were significant differences in villi length between the PbA group and the PbA+Lmu group (P < 0.0001) and the PbA+GABA group (P < 0.0001). The villi length of the PbA+Lmu group was shorter than that of the PbA+GABA group, but there was no significant difference between the two groups (P = 0.343). Compared with the DHA group, the intestinal villi length of the DHA+Lmu group (P = 0.004) and the DHA+GABA group (P = 0.002) was significantly increased. There was no significant difference between the DHA+Lmu group and the DHA+GABA group (P = 0.721).

[0101] like Figure 8As shown in Figure B, intestinal crypt depth was quantitatively analyzed. Compared with the control group, the crypt depth of all ECM mice groups was shortened. Crypt depths were significantly different between the PbA group and the PbA+Lmu group (P<0.0001) and the PbA+GABA group (P<0.0001). Crypt depths in the PbA+Lmu group were smaller than those in the PbA+GABA group, but there was no significant difference between the two groups (P=0.895). Crypt depths were slightly smaller in the DHA+Lmu group (P=0.25) and the DHA+GABA group (P=0.55) than in the DHA group. There was no significant difference between the DHA+Lmu group and the DHA+GABA group (P=0.737).

[0102] like Figure 8 As shown in Figure C, the number of goblet cells in PAS-stained intestinal pathological sections was analyzed. The mean number of goblet cells in the PbA + Lmu group was slightly higher than that in the PbA group, but the difference between the two groups was not statistically significant (P = 0.470). The mean number of epithelial cells in the DHA + Lmu group was significantly higher than that in the DHA group, but the difference between the two groups was not statistically significant (P = 0.81). The mean number of epithelial cells in the DHA + GABA group was higher than that in the DHA group, but the difference between the two groups was not statistically significant (P = 0.468). The mean number of goblet cells in the DHA + Lmu group was lower than that in the DHA + GABA group, but the difference between the two groups was not statistically significant (P = 0.642).

[0103] like Figure 8 As shown in Figure D, intestinal permeability of mice was analyzed using FITC-dextran. In the PbA group, FITC significantly leaked from the intestinal lumen into the blood. Compared with the PbA+Lmu group, the PbA group had the least difference in vascular leakage (P=0.004), and compared with the PbA+GABA group, the PbA group had the greatest difference in vascular leakage (P=0.002). There was no significant FITC leakage in the DHA-treated group, a small amount in the DHA+GABA group, and no significant FITC leakage in the DHA+Lmu group.

[0104] like Figure 9As shown, compared with intestinal histological analysis of uninfected mice, the DHA+GABA group showed the most intact intestinal structure among all treatment groups, with clear and even distribution of intestinal epithelial cells. The PbA group showed the most severe intestinal structural damage and the most pronounced inflammatory response, with shortened and thickened villi, desquamated intestinal epithelial cells, and microscopic bleeding. The number of goblet cells embedded in the mucosa was significantly reduced, as was the amount of mucin secreted into the intestinal lumen, and the optical density of goblet cells was significantly decreased. The PbA+Lmu and PbA+GABA groups showed loosely arranged small intestinal villi and severe damage to villus structure, but the degree of reduction in villus length and crypt depth was significantly less than that in the PbA group. Compared with the DHA group, the DHA+Lmu (P=0.004) and DHA+GABA (P=0.002) groups showed a stronger protective effect on small intestinal structure, with denser villi arrangement and significantly increased villus length. Compared with the DHA group, the crypt depths of the DHA+Lmu group (P=0.25) and the DHA+GABA group (P=0.55) were slightly smaller. The DHA+GABA and DHA+Lmu groups showed normal intestinal structure, with closely arranged villi and clear borders, significantly increased villus length and crypt depth, and a significant increase in goblet cell number, suggesting improved intestinal barrier function.

[0105] The blood-brain barrier (BBB) ​​is formed by brain microvascular endothelial cells lining the walls of brain capillaries. In the PbA group, malaria parasites were confined to brain vascular erythrocytes, and iRBCs adhered to brain vascular endothelial cells, leading to the destruction of the blood-brain barrier. Figure 10 As shown in Figure A, the DHA+Lmu group effectively reduced the aggregation of inflammatory cells and the separation of iRBCs in brain capillaries. Compared with the control group, iRBCs and leukocytes aggregated in the lumen of PbA mice, forming a rosette effect. iRBCs adhered to brain endothelial cells, blocking cerebral blood vessels, leading to vasoconstriction, spasm, and damage, with a small number of scattered bleeding spots appearing around the lumen. In the DHA group, a small amount of leukocytes still blocked the lumen of the blood vessels. The incidence of cerebral vascular obstruction and hemorrhage in the DHA+Lmu and DHA+GABA groups was lower than that in the DHA group. There was almost no cerebrovascular obstruction and leukocyte infiltration, which significantly improved cerebral vascular damage and had a strong protective effect on brain tissue.

[0106] Histological analysis of livers from uninfected mice served as controls. Figure 10As shown in Figure B, among all treatment groups, the DHA+GABA group had the most complete hepatic lobule structure and the strongest protective effect on the liver. The PbA group had the most severe damage to the hepatic lobule structure, with enlarged and twisted central veins and darkly stained hepatocyte cytoplasm due to lack of glycogen. A large number of iRBCs and inflammatory cells were retained in the central hepatic veins, leading to acute liver damage. In the PbA+Lmu group, the central hepatic veins and hepatic veins were enlarged and congested, the hepatic plates were irregularly arranged, the cell cytoplasm was vacuolated, the hepatic sinusoids were dilated, a large number of white blood cells blocked the veins, and malarial pigment deposition was obvious. The hepatic plates and sinusoids were disorganized, the hepatic lobules were not arranged radially, the hepatic cell cytoplasm was darkly stained, but malarial pigment deposition was significantly reduced, which was significantly reduced compared with the PbA group. Compared with the mild liver damage in the DHA group, the DHA+Lmu group (P=0.0011) and the DHA+GABA group (P=0.003) had a stronger protective effect on the liver lobule structure, with liver cells arranged more densely and radially around the central vein. White blood cell recruitment was significantly reduced, the liver lobule structure returned to normal, and there was less hemochromatin deposition, indicating a better ability to protect the liver.

[0107] like Figure 10 As shown in Figure C, spleen pathological analysis showed that the RePu area was significantly widened and the WhPu area was reduced in the PbA, PbA+Lmu, PbA+GABA, and DHA groups, indicating severe anemia and increased extramedullary hematopoiesis. Compared with the above four groups, the RePu area was smaller in the DHA+Lmu and DHA+GABA treatment groups, suggesting improved anemia. The WhPu area was larger, and the spleen structure was similar to that of the control group. In terms of RePu area, there was little difference in the damaged spleen structure between the DHA+Lmu and DHA+GABA groups. The RePu width in the DHA+GABA group was smaller than that in the DHA+Lmu group. Compared with DHA alone, the RePu area was significantly smaller in the DHA+Lmu (P<0.001) and DHA+GABA (P<0.001) groups, indicating reduced extramedullary hematopoiesis. Therefore, combined bacterial and drug treatment is more effective in spleen disease than DHA monotherapy.

[0108] 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. Application of GABA in the preparation of drugs for improving and treating cerebral malaria in mice caused by Plasmodium berghei; The treatment of mouse cerebral malaria comprises one or more of the following: A) Improve mouse behavior; B) Improve immune pathological damage to various organs; C) reduce parasitemia; D) Prolong the survival of mice.

2. The use according to claim 1, characterized in that The drug further comprises one or three of dihydroartemisinin, rapamycin and atorvastatin.

3. The use according to claim 1, characterized in that The drug must contain dihydroartemisinin.

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