Application of GABA (gamma-aminobutyric acid) driven by lactobacillus rhamnosus in treatment of cerebral malaria of mice

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of GABA (gamma-aminobutyric acid) driven by lactobacillus rhamnosus to treatment of cerebral malaria of mice. According to the invention, the obviously enriched lactobacillus muricatum is found, and the lactobacillus muricatum is proved to be capable of generating GABA (gamma-aminobutyric acid). Afterwards, on the mouse cerebral malaria model, under the condition that the lactobacillus rhamnosus and the GABA are independently used or auxiliary dihydroartemisinin is used, the effect of the lactobacillus rhamnosus and the GABA on improving and treating the mouse cerebral malaria is evaluated. Results show that the strain and the metabolite GABA thereof can effectively improve behavioristics of mice and immune pathological injuries of all organs, reduce protozoemia and prolong the lifetime of the mice whether the strain is independently used or combined with dihydroartemisinin, and can effectively treat cerebral malaria of the mice compared with the strain which is independently used with drugs. The invention provides a brand-new solution for treating severe malaria, especially human brain malaria, caused by malignant malaria from the perspective that the probiotics drive metabolites of the probiotics to assist the anti-malarial drug in treating rat brain malaria.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of Lactobacillus murinus driving GABA in treating mouse cerebral malaria. Background Art

[0002] Cerebral malaria (CM) is a neurological term published in 2020. It is a parasitic disease of the brain caused by direct invasion of malarial parasites, especially Plasmodium falciparum. It is a serious disease with a high mortality rate and is one of the most serious types of malaria. About 1% to 2% of falciparum malaria will eventually develop into cerebral malaria, which mostly occurs in children under 5 years old in Africa, with a mortality rate of about 15% to 20%. Clinically, cerebral malaria may present with mild hemiparesis, convulsions, ataxia, coma and impaired consciousness, meningeal irritation signs and even death. Even for patients who receive early standard antimalarial chemotherapy drugs, there is still a very high mortality rate after the malarial parasites are cleared from the body. In addition, about 25% of survivors will have sequelae of neurological complications and cognitive dysfunction. 10% to 20% of surviving children have persistent neurological deficits, cognitive dysfunction, behavioral disorders, and motor impairment.

[0003] Currently, quinine and artemisinin are recommended drugs for the treatment of CM. Although artemisinin drugs have a highly effective antimalarial effect, the dead parasites after killing the malarial parasites still block the local microvessels. The local ischemic changes and microvascular damage caused by the blockage cannot be effectively repaired, making the mortality rate of cerebral malaria in children and adults as high as 18% and 30%. Studies have also shown that artemisinin-based combination therapies (ACTs) often fail to protect against cell death, neural damage, and cognitive deficits. Therefore, artemisinin drugs alone are not enough to prevent death or neurological dysfunction in all patients with cerebral malaria. This suggests that we need to continue to explore new artemisinin-based drug combinations, treatment options, and adjuvant treatments based on existing treatment strategies.

[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 the number of harmful bacteria increased. Subsequently, based on previous studies, we continued to explore the effects and functions of different drug combinations of dihydroartemisinin (DHA), rapamycin (RAP) and atorvastatin (AVA) on intestinal flora in mouse cerebral malaria models. The results showed that Lactobacillus murinus was significantly enriched after the use of dihydroartemisinin. However, it is not clear whether the bacteria 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 nervous system diseases. However, the role of intestinal probiotics, especially Lactobacillus murinus, in infectious diseases, especially cerebral malaria, has not been reported, and how it works. The reason for the lack of the above 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 are some progress in the above, but there is still a certain distance from actual clinical treatment. The present invention is prompted by the intestinal flora treatment of major diseases including tumors, and the study of the interaction between intestinal flora and parasitic infection was carried out earlier in China.

[0006] To this end, the present invention attempts to reveal the role of Lactobacillus murinus in the treatment of mouse cerebral malaria and its possible mechanism of action. Specifically, a mouse cerebral malaria model was first established, and then the bacteria or GABA combined with dihydroartemisinin or the bacteria or GABA alone were used for treatment on the model, and the therapeutic effect of the bacteria on mouse cerebral malaria was observed through behavioral, pathological, molecular biological and other technical means. Summary of the invention

[0007] The present invention first provides an application of Lactobacillus murinus driving GABA in treating mouse cerebral malaria, 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 the immune pathological damage of 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 the immune pathological damage of 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 contain 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 finds significantly enriched Lactobacillus murinus and metabolite GABA through combined analysis of intestinal flora and metabolomics. Subsequently, we confirmed that Lactobacillus murinus can produce GABA. Subsequently, we evaluated the effect of improving and treating mouse cerebral malaria in the mouse cerebral malaria model by using Lactobacillus murinus, GABA alone or with the aid of dihydroartemisinin. The results suggest that whether used alone or in combination with dihydroartemisinin, the bacteria and its metabolite GABA can effectively improve mouse behavior, immune pathological damage of various organs, reduce parasitemia, and prolong the survival of mice. Compared with the use of drugs alone, it can effectively treat mouse cerebral malaria. Since the Lactobacillus murinus used is a common probiotic lactobacillus, dihydroartemisinin is a clinical first-line antimalarial drug. If it is clinically transformed for the treatment of human cerebral malaria, the conversion time can be shortened. The present invention is based on the concept that the metabolites produced by probiotics can assist drugs in the treatment of malaria, and from this perspective, it provides a solution for the treatment of human cerebral malaria. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Experimental groups and treatment scheme. C57BL / 6 mice were infected with PbA to establish the ECM model, and the day of infection was day 0. Drug treatment was given on the 3rd day after infection, and L. Murinus and GABA were given by gavage on day 0 after infection. The 3rd to 7th day after infection was the ECM stage of bacterial and drug synergistic 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 the 9th day after infection and the histopathological observation and intestinal barrier integrity assessment on the 10th day.

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

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

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

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

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

[0035] Figure 7 Semi-quantitative analysis of hemochromatin and red pulp area. (A) Analysis of hemochromatin ratio in liver of different bacterial drug treatment groups; (B) Analysis of hemochromatin ratio in spleen of different treatment groups; (C) Quantitative analysis of red pulp (RePu) area of ​​different treatment groups. The statistical analysis method was one-way ANOVA, and each one-way ANOVA used LSD post hoc. Error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0036] Figure 8 Related intestinal indicators 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. The statistical analysis method was one-way ANOVA, and each one-way ANOVA was tested with LSD post hoc. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0037] Fig. 9 HE staining of intestinal tissue (100×). The orange rectangle indicates that the villus structure of the small intestine is destroyed and the goblet cells are missing. The red arrow indicates the separation of intestinal epithelial cells. Scale bar: 50μm.

[0038] Fig.10Histopathological observation of mouse brain, liver, and spleen. (A) Hemoglobin-eosin (HE) staining of the brain (magnification, ×1,000). The purple arrows point to red blood cells (RBC), the black arrows point to brain microvascular endothelial cells (BMEC), the green oval boxes represent iRBC and leukocyte aggregation to form rosettes, the red rectangular boxes represent leukocyte obstruction, and the yellow oval boxes represent hemorrhage. Scale bar: 10 μm. (B) HE staining of the liver (magnification, ×400). The red arrows represent normal red blood cells in the venous vessels, the black arrows represent iRBC, the yellow rectangular boxes represent hemoglobin, the red rectangular boxes represent fat vacuoles, and the blue oval circles represent the adhesion and aggregation of leukocytes and iRBC. Scale bar: 20 μm. (C) HE staining of the spleen (magnification, ×400). The red area represents RePu, and the blue area represents 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 more clear, 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 strains: Female C57BL / 6 mice (weight 18-22 g, 8-10 weeks old) were purchased from HNSJA Co., Ltd., Changsha, China. They were raised under specific pathogen-free conditions and fed with UV-irradiated feed and purified water to maintain appropriate living and feeding standard experimental conditions (25±3℃). All mice were acclimatized to the environment 1 week before the experiment.

[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, Bei Xi Man OLYMMPUS Microscope BX53 Olympus Corporation, Japan Centrifuge 5424R Eppendorf, Germany Constant temperature metal bath MK-20 China, Aosheng Tissue Slice Image Imaging System BX53 Olympus Corporation, Japan Micropipette 2-volume pipette us imaging system Eppendorf, Germany Multifunctional ELISA reader SYNERGY-HT BioTek, USA Mouse tail vein injection instrument KW-XXY Calvin, China 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 Dihydroartemisinin China, Myrel Rapamycin China, Myrel Atorvastatin Solarbio, China syringe China, Weigao Cryotubes Thermo Fisher Scientific, USA Glass slides China, Shitai Methanol China, Zhongtian Dimethyl sulfoxide VWR, USA EvansBlue SIGMA, Germany Formamide SIGMA, Germany 0.9% saline Kelun, China Universal tissue fixative China, Absin Slide Storage Box China, Blue Sky PBS buffer powder Servicebio, UK Pipette Tips Thermo Fisher Scientific, USA

[0055] Example 2

[0056] (1) Resuscitation and passage: Place the PbA parasite strain frozen in liquid nitrogen in a 37°C water bath. After thawing, draw blood with a 1 ml syringe and immediately inoculate it intraperitoneally into C57BL / 6 mice at a dose of 0.2 ml / mouse. This is blood seed resuscitation. When the parasitemia level of the blood seed mice reaches 15%-30%, remove the eyeballs and draw blood in a heparin anticoagulant tube. 6 The iRBCs-infected erythrocytes were passaged into offspring C57BL / 6 mice at a dose of 0.2 ml / mouse.

[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 mining, as follows: 30 female C57BL / 6 mice were randomly divided into 3 groups: infection-untreated group (PbA), DHA treatment group (DHA), and DHA combined with RAP and AVA treatment group (DHA.RAP.AVA).

[0059] 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 third day after infection, the PbA group was intraperitoneally injected with 5% DMSO as a control. The DHA group was given 3 mg / kg DHA. The DHA.RAP.AVA group was given 5 mg / kg RAP, 3 mg / kg DHA and 40 mg / kg AVA. The above mice were treated with drugs by intraperitoneal injection for 5 consecutive days.

[0060] (2.2) Sample collection and sequencing analysis: On the 8th day after infection, fecal pellets from each group of mice were collected with sterile tweezers, placed in sterile centrifuge tubes, immediately frozen in liquid nitrogen, and stored in a -80°C refrigerator until use. At the same time, mouse brain tissue was collected for metabolomics sequencing. Subsequently, the intestinal flora sequencing results were jointly analyzed with brain metabolites.

[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 containing 0.5 g / L L-cysteine, and cultured anaerobically at 37°C for 24 h. The bacteria cultured in MRS (5%, V / V) were inoculated into CM medium and cultured anaerobically at 37°C for 48 h. After incubation, the bacteria were harvested by centrifugation, resuspended in suspension for standby use (skim milk 50 g / L; lactose 30 g / L; yeast extract 50 g / L; ascorbic acid, 50 g / L), and freeze-dried at -80°C for storage. At the same time, 100 μL of bacterial solution was taken, centrifuged at 3000 g for 5 min, resuspended in 20 μL PBS, and 10 μL was taken for microscopic examination with Gram stain.

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

[0065] L. murinus was cultured as before. A certain amount of bacterial solution was taken and the GABA content in the bacteria was detected according to the instructions of the "Bacterial γ-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, namely, 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 days 0 and 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, and 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 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] Protozoaemia observation: Blood was collected from the tail vein to prepare thin blood smears, which were then stained with Giemsa. Protozoaemia was evaluated by Giemsa-stained thin blood smears under a 100x optical microscope. Protozoaemia was examined and quantified by counting the number of iRBCs in at least 1,000 red blood cells.

[0071] 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 was evaluated using 10 parameters (hair, limb strength, defense ability, auricular reflex, toe reflex, contact reflex, gait, balance, body posture and exploratory activity). If mice gradually develop characteristics such as unsteady walking, ataxia, messy hair, no extension, loss of toe reflex, loss of auricular reflex, convulsion, coma, and even death, ECM was successfully established. Parasitemia was evaluated and quantified by counting the number of iRBCs in 1,000 red blood cells by thin blood film smear under a 100× optical microscope.

[0072] Assessment of blood-brain barrier integrity: The protection of the brain was assessed by the integrity of the blood-brain barrier (BBB). 1% EB dye was diluted in 0.9% NaCl. On the 8th day after infection, 200 μl of 1% EB dye solution was injected into the tail vein of mice, the dye was allowed to circulate for 30 min, and then the mice were 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 min, and the absorbance was measured at 630 nm using an enzyme reader to quantify EB according to the standard curve.

[0073] Histopathological observation: After the end of administration, i.e., on the 8th day after infection, tissue samples were collected. After euthanasia, brain, liver, spleen, and small intestine tissues were immediately collected and washed three times in cold PBS to remove blood. Then, they were fixed with a universal tissue fixative for 24 hours and embedded in paraffin. Continuous 4 μm thick sections were cut and HE stained to observe microvascular obstruction and leakage. The sections were observed using an optical microscope, and Olympus cellSens standard 1.13 software was used for image acquisition.

[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, DHA monotherapy group, and 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 tissues were collected for metabolomics sequencing. Figure 3 As shown in the figure, brain metabolome analysis found that the content of GABA in the untreated group, DHA monotherapy group, and DHA.RAP.AVA group also showed an increasing trend (indicated by the red arrow), suggesting that this metabolite plays an active 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 alone 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 internal mechanism behind the positive correlation needs further confirmation.

[0081] Example 3 Observation on the effect of lactic acid and its metabolite GABA on the treatment of cerebral malaria in mice

[0082] (1) The microscopic morphology of Lactobacillus murinus is shown below: Figure 5 shown.

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

[0084] The test results showed that the GABA content in the bacteria was 2.595mmol / mL and 2.251mmol / mL at 0 and 10min, respectively. This is consistent with the GABA content produced by other lactobacilli reported in the literature. The test results suggest that Lactobacillus murinus can preliminarily 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 treatment and Lmu intervention in the 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 the 9th day, respectively. On the 10th day, 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 (only GABA intervention in the mouse cerebral malaria model) and the DHA group (only DHA treatment in the mouse cerebral malaria model), the average body weight of the mice in the DHA+GABA group (DHA+GABA group-PbA+GABA group) / PbA+GABA group) and the DHA group (only DHA treatment in the mouse cerebral malaria model) increased by 28.6% [(DHA+GABA group-PbA+GABA group) / PbA+GABA group] and 3.16% [(DHA+GABA group-DHA group) / DHA group] on the 9th day. On the 7th day, the RMCBS scores of the 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 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 index 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 as a benchmark reference, where D8, D9 and D10 represent the days after infection.

[0091] like Figure 6 As shown in A, from day 0 after infection, the weight changes of mice in all drug-treated groups showed a general trend of first decreasing, then increasing, and then decreasing compared with the non-drug-treated group. The average weight of the PbA+Lmu group was higher than that of the PbA+GABA group, but the difference was not statistically significant. The average weight of the DHA+Lmu group was higher than that of the DHA group, and there was a significant difference between the two groups (P=0.0006). The average 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 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). In the combined bacterial and drug treatment group, the weight of the DHA+Lmu group decreased slightly, but the decrease was the smallest, while the weight of the DHA group decreased the most, and the difference between the two was statistically significant (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 B, the RMCBS scores of mice in each group showed a downward trend. The mice in the PbA group began to show symptoms of decreased exploratory ability, weakened limb strength and unstable walking on the 4th day after infection, and the RMCBS score decreased rapidly with the appearance of CM symptoms. On the 7th day after infection, the RMCBS score of mice in the PbA group was less than 4 points, which was consistent with the symptoms shown by the mice, namely reduced exploratory behavior, weakened reflexes, reduced self-defense ability, coma and convulsions. On the 10th day after infection, the RMCBS score of mice was only 1.8, while the RMCBS scores of mice in the PbA+Lmu group and PbA+GABA group were significantly higher during the same period. The RMCBS scores of mice in the drug treatment group decreased slightly from the 4th to the 7th day after infection, but they all remained within the normal range, and there was no statistically significant difference among the three groups. In the late stage of infection, the scores of the DHA group decreased faster than those of the DHA+Lmu group and the DHA+GABA group. On the 16th day after infection, the RMCBS score of the DHA+Lmu group was significantly higher than that of the DHA group (P=0.003). There was no 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 6The results of peripheral blood parasitemia test shown in C showed that the parasitemia levels in each group showed an upward trend and reached the initial peak in the first 3 days of infection. The parasitemia level in the PbA group began to rise rapidly from 2 days after infection and peaked on the 8th day after infection, showing a typical parasite growth pattern. In contrast, the parasitemia of mice treated with drugs decreased between the initial peak and the later proliferation period. On the 8th day after infection, the parasitemia of the PbA+Lmu group (P=0.068) and the PbA+GABA group (P=0.050) were lower than that of the PbA group, and the difference was statistically significant. The parasitemia level of the DHA+GABA group was significantly lower than that of the DHA group, and the difference was statistically significant (P=0.024). The parasitemia level of the DHA+Lmu group was slightly lower than that of the DHA group, and there was no statistically significant difference between the two groups (P=0.181). There was no statistically significant difference between the DHA+GABA group and the DHA+Lmu group (P=0.217).

[0094] like Figure 6 As shown in D, mice infected with PbA developed neurological symptoms on day 4 after infection, leading to death on days 5-10 after infection. About 50% of mice died of ECM within 8 days after infection, and the parasitemia rate reached 48.96-56.73%. All mice in the PbA group developed typical symptoms of ECM and died within 10 days after infection. The survival rate of the PbA+Lmu group was significantly higher than that of the PbA group, and the difference was statistically significant (P=0.016). The survival time of the PbA+Lmu group was higher than that of the PbA+GABA group (P=0.005). The average survival days of the PbA group (8.10±2.12) were higher than those 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 within 18 days. There was no significant difference in survival rate between the DHA group and the DHA+Lmu group (P=0.607), between the DHA+GABA group and the DHA group (P=0.213), and between the DHA+Lmu group and the DHA+GABA group (P=0.223).

[0095] exist Figure 7In the DHA+Lmu group (using both DHA treatment and Lmu intervention in the mouse cerebral malaria model), compared with the PbA+Lmu group (using only Lmu intervention in the mouse cerebral malaria model) and the DHA group (using only DHA treatment in the mouse cerebral malaria model), the hemochromatin in the mouse liver 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 in the mouse spleen 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 PbA+GABA group (only GABA intervention in the mouse cerebral malaria model) and the DHA group (only DHA treatment in the mouse cerebral malaria model), the DHA+GABA group (DHA treatment in the mouse cerebral malaria model) showed that the hemochromatin in the liver of 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 in the spleen of 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 hemochromatin in the pathological sections of liver tissue stained with HE. The effect of PbA combined with L.murinus in clearing hepatic hemochromatin was significantly better than that of the PbA group, and the difference between the two groups was significant (P < 0.0001). There was no significant difference in the proportion of hepatic hemochromatin between the PbA + Lmu group and the PbA + GABA group (P = 0.244). The ability of DHA combined with L.murinus to clear hemochromatin was significantly better than that of the DHA group, and the difference between the two groups was significant (P = 0.0011). The ability of DHA combined with GABA to metabolize hemochromatin was significantly better than that of the DHA group, and the difference between the two groups was significant (P = 0003). There was no statistically significant difference in hemochromatin between the DHA + Lmu group and the DHA + GABA group (P = 0.306).

[0098] Figure 7 B is the quantitative analysis of the proportion of hemochromatin in spleen tissue. The ability of PbA+Lmu group (P<0.0001) and PbA+GABA group (P<0.0001) to remove hemochromatin was stronger than that of PbA group. The proportion of hemochromatin in PbA+Lmu group was higher than that in PbA+GABA group, but the difference between the two groups was not statistically significant (P=0.098). Compared with DHA group, the proportion of hemochromatin in DHA+Lmu group (P=0.003) and DHA+GABA group (P=0.006) was lower. There was no statistically significant difference between DHA+Lmu group and DHA+GABA group (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 of ​​the PbA+GABA group was smaller than that of the PbA+Lmu group, but there was no significant difference between the two (P=0.222). Compared with the DHA group, the RePu area of ​​the DHA+Lmu group (P=0.0002) and the DHA+GABA group (P=0.0002) was significantly reduced. There was also a significant difference between the DHA+Lmu group and the DHA+GABA group (P=0.016). These results indicate that compared with the DHA group, the DHA+Lmu group and the DHA+GABA group have more effective protective effects on the spleen.

[0100] like Figure 8 As shown, the length of HE-stained villi was quantitatively analyzed. Compared with the control group, the villi length of each group of ECM mice was shortened. There was a significant difference in the 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 B, the intestinal crypt depth was quantitatively analyzed. The crypt depth of each group of ECM mice was shortened compared with the control group. There was a significant difference in the crypt depth between the PbA group and the PbA+Lmu group (P<0.0001) and the PbA+GABA group (P<0.0001). The crypt depth of the PbA+Lmu group was smaller than that of the PbA+GABA group, but there was no significant difference between the two groups (P=0.895). Compared with the DHA group, the crypt depth of the DHA+Lmu group (P=0.25) and the DHA+GABA group (P=0.55) was slightly smaller. 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 C, the number of goblet cells in the intestinal pathological sections stained by PAS was analyzed. The average number of goblet cells in the PbA+Lmu group was slightly higher than that in the PbA group, but there was no significant difference between the two groups (P=0.470). The average number of epithelial cells in the DHA+Lmu group was significantly higher than that in the DHA group, but there was no significant difference between the two groups (P=0.81). The average number of epithelial cells in the DHA+GABA group was higher than that in the DHA group, but there was no significant difference between the two groups (P=0.468). The average number of goblet cells in the DHA+Lmu group was less than that in the DHA+GABA group, but there was no significant difference between the two groups (P=0.642).

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

[0104] like Fig. 9As shown in the figure, compared with the results of intestinal histological analysis of uninfected mice, among all treatment groups, the intestinal structure of mice in the DHA+GABA group was the most complete, and the distribution of intestinal epithelial cells was clear and uniform. The intestinal structure of the PbA group was the most severely damaged, the inflammatory reaction was the most obvious, the villi were shortened and thickened, the intestinal epithelial cells were shed, bleeding was visible under the microscope, the number of goblet cells embedded in the mucosa was significantly reduced, the amount of mucin secreted into the intestinal lumen was also reduced, and the optical density of goblet cells was significantly reduced. The small intestinal villi of the PbA+Lmu group and the PbA+GABA group were loosely arranged, and the villi structure was severely damaged, but the shortening of the villi length and crypt depth was significantly lower than that of the PbA group. Compared with the DHA group, the DHA+Lmu group (P=0.004) and the DHA+GABA group (P=0.002) had a stronger protective effect on the small intestinal structure, the villi were more tightly arranged, and the villi length was significantly increased. Compared with the DHA group, the crypt depth of the DHA+Lmu group (P=0.25) and the DHA+GABA group (P=0.55) was slightly smaller. The intestinal structure of the DHA+GABA group and the DHA+Lmu group was normal, the intestinal villi were closely arranged, the boundaries were clear, the villus length and crypt depth were significantly increased, and the number of goblet cells was significantly increased, which can improve the 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, Plasmodium was confined to brain vascular erythrocytes, and iRBCs adhered to brain vascular endothelial cells, leading to the destruction of the blood-brain barrier. Fig.10 As shown in 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 in the lumen of mice in the PbA group aggregated together to form a rosette effect, and iRBCs adhered to brain endothelial cells, blocking cerebral blood vessels, causing cerebral vasoconstriction, spasm and damage, and a small number of scattered bleeding spots appeared around the lumen blood vessels. In the DHA group, there was still a small amount of leukocyte blockage in the lumen of the blood vessels. The incidence of cerebral vascular obstruction and hemorrhage in the DHA+Lmu group and the DHA+GABA group was lower than that in the DHA group, and there was almost no obstruction and leukocyte infiltration in the cerebral blood vessels, 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. Fig.10As shown in B, among all the treatment groups, the DHA+GABA group had the most complete liver lobule structure and the strongest protective effect on the liver. The PbA group had the most severe damage to the liver lobule structure, the central vein was enlarged and twisted, and the hepatocyte cytoplasm was darkly stained 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 vein and hepatic vein were enlarged and congested, the hepatic plates were arranged irregularly, 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 the malarial parasite 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, the hepatocytes were arranged more tightly and radially around the central vein, the recruitment of leukocytes was significantly reduced, the liver lobule structure returned to normal, and there was less malarial pigment deposition, which had a better ability to protect the liver.

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

Claims

1. The application of Lactobacillus murinus driving GABA in treating cerebral malaria in mice, characterized in that: The application is in vivo or in vitro; Optionally, the treatment of mouse cerebral malaria comprises one or more of the following: A) Improve mouse behavior; B) Improve the immune pathological damage of various organs; C) reduce parasitemia; D) prolong the survival of mice.

2. The use according to claim 1, characterized in that: The in vitro is bacteria, and the in vivo is mice.

3. The use of GABA in treating cerebral malaria in mice, characterized in that: The application is in vivo; Optionally, the treatment of mouse cerebral malaria comprises one or more of the following: A) Improve mouse behavior; B) Improve the immune pathological damage of various organs; C) reduce parasitemia; D) prolong the survival of mice. Optionally, the GABA is prepared by Lactobacillus murinus or purchased from a reagent company (the source is convenient, the extraction of GABA from Lactobacillus murinus is complicated, so it is purchased from a company for time-saving and convenient operation).

4. The use of Lactobacillus murinus or GABA in the preparation of a drug for improving and treating cerebral malaria caused by Plasmodium berghei in treating cerebral malaria in mice.

5. The use according to claim 4, characterized in that: The mouse cerebral malaria is cerebral malaria caused by Plasmodium berghei.

6. The use according to claim 5, characterized in that: The drug also contains one or three of dihydroartemisinin, rapamycin and atorvastatin.

7. The use according to claim 6, characterized in that: The drug must contain dihydroartemisinin.

8. A pharmaceutical composition for treating mouse cerebral malaria, characterized in that: The medicament comprises Lactobacillus murinus and or GABA.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also comprises dihydroartemisinin, rapamycin and atorvastatin.

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