Application of cGAMP in prevention of cerebral malaria

Abstract

The embodiment of the invention discloses application of cGAMP in prevention of cerebral malaria, and belongs to the technical field of prevention and treatment of zoonosis parasitic diseases. CGAMP is used as a second messenger in the cell, responds to cytoplasm double-stranded DNA synthesis, activates an innate immune STING pathway, the cGAMP is combined with STING, and the STING is collected and phosphorylated, so that further collection and activation of TANK binding kinase 1 (TBK1) are promoted, combination and phosphorylation of interferon regulatory factor 3 (IRF3) are caused, the activated IRF3 stimulates expression of I-type interferon (IFN), and the IFN plays a key role in antivirus and innate immune response, and can be used for preventing and treating the virus and the innate immune response. The IFN-I (IFN-alpha and IFN-beta) plays an important role in plasmodium infection, so that the IFN-I (IFN-alpha and IFN-beta) plays a key recognition role in the plasmodium infection process, cerebral malaria can be prevented, and the life quality of a patient is greatly improved.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of prevention and treatment of zoonotic parasitic diseases, and specifically to the application of cGAMP in preventing cerebral malaria. Background Art

[0002] Malaria is transmitted by the bites of female Anopheles mosquitoes infected with Plasmodium. Among all the currently known human malaria parasites, Plasmodium vivax and Plasmodium falciparum are the most pathogenic malarial parasites. The early symptoms of malaria are fever, headache and chills. Most malaria-infected people clinically present with asymptomatic parasitemia. As the disease progresses, it can induce a variety of disease syndromes (severe malaria), including severe anemia, metabolic acidosis and neurological dysfunction. Among them, human cerebral malaria syndrome (HCM) is a rare but life-threatening complication of severe malaria, manifested as acute cerebrovascular encephalopathy. Cerebral malaria is mainly related to Plasmodium falciparum infection, and the incidence of cerebral malaria in children under 5 years old is higher than that in adults. Mouse models and patient data show that parasite replication in blood vessels leads to immune-mediated vascular neuropathology, and the resulting dysfunction of the blood-brain barrier leads to vasogenic cerebral edema, increased intracranial pressure, brain herniation and global ischemia. Despite treatment with effective antiparasitic drugs, 20% of patients with cerebral malaria die from the disease, and many cerebral malaria survivors suffer from neurocognitive impairment. Therefore, the development of preventive, adjunctive, and neuroprotective treatments for cerebral malaria is of vital importance.

[0003] The cGAS-STING pathway is a central cell cytoplasmic DNA sensor that can recognize exogenous DNA introduced by pathogen invasion, induce the secretion of type I interferon and the production of inflammatory cytokines, activate the body's immune system, and resist the invasion of pathogenic microorganisms. Cyclic GMP-AMP synthase (cGAS) contains a nucleotidyl transferase domain and two DNA binding domains. When dsDNA is present, cGAS will bind to dsDNA and catalyze the cyclization of ATP and GTP to form endogenous cyclic dinucleotide (CDN) compounds, such as 2'3'-cGAMP, 2'2'-cGAMP, 3'3'-cGAMP, c-diAMP, etc. The stimulator of interferon genes (STING, also known as TMEM173) is a downstream receptor protein of CDNs. After binding to CDNs, it forms a multimeric complex. The multimeric STING detaches from the endoplasmic reticulum and translocates to the Golgi apparatus, recruiting TBK1 and IRF3, thereby promoting the transcriptional expression of type I interferon (IFN) genes. Studies have shown that Plasmodium falciparum activates the STING-dependent cytoplasmic DNA sensing pathway by secreting an extracellular vesicle (EV) containing non-coding RNA and genomic DNA into the host cytoplasm, so STING plays a key recognition role in the process of Plasmodium infection. At present, no research has reported the use of cGAMP to treat malaria or prevent the occurrence of cerebral malaria. Summary of the invention

[0004] To this end, an embodiment of the present invention provides an application of cGAMP in preventing cerebral malaria, so as to solve the problem that there is no attempt to use cGAMP to treat malaria or prevent the occurrence of cerebral malaria in the prior art.

[0005] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of an embodiment of the present invention, the present invention provides use of cGAMP as a STING agonist in the preparation of a drug for preventing cerebral malaria.

[0007] Furthermore, the cGAMP is 3'3'-cGAMP.

[0008] Furthermore, the prevention of cerebral malaria is to reduce the mortality rate of cerebral hemorrhage caused by Plasmodium infection.

[0009] Furthermore, cGAMP was diluted with saline and injected into the tail vein of infected mice.

[0010] According to a first aspect of an embodiment of the present invention, the present invention provides use of cGAMP in combination with a TLR9 agonist in the preparation of a drug for preventing cerebral malaria.

[0011] Furthermore, the TLR9 agonist is ODN 1826sodium.

[0012] The embodiments of the present invention have the following advantages:

[0013] As a second messenger in the cell, cGAMP responds to the synthesis of cytoplasmic double-stranded DNA and activates the innate immune STING pathway. cGAMP binds to STING, recruits STING and phosphorylates it, thereby promoting the further recruitment and activation of TANK binding kinase 1 (TBK1), leading to the binding and phosphorylation of interferon regulatory factor 3 (IRF3). Activated IRF3 stimulates the expression of type I interferon (IFN). IFN plays a key role in antiviral and innate immune responses. IFN-I (IFN-α and IFN-β) plays an important role in malarial parasite infection, thus playing a key recognition role in the process of malarial parasite infection, which can prevent cerebral malaria and significantly improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0015] Figure 1 A comparison chart of the survival rates of experimental cerebral malaria model mice after injection of cGAMP or saline provided in Example 1 of the present invention;

[0016] Figure 2 This is a comparison chart of infection rates in experimental cerebral malaria model mice after injection of cGAMP or normal saline provided in Example 1 of the present invention;

[0017] Figure 3 A comparison chart of the survival rates of non-experimental cerebral malaria model mice injected with cGAMP or saline provided in Example 1 of the present invention;

[0018] Figure 4 A comparison chart of the survival rates of the experimental cerebral malaria mouse model provided in Example 1 of the present invention after injection of cGAMP at different times;

[0019] Figure 5(A) Fluorescence image of the WT group after the experimental cerebral malaria model mice were injected with cGAMP or saline provided in Example 1 of the present invention; (B) Fluorescence image of the drug-treated group; (C) Fluorescence intensity comparison graph;

[0020] Figure 6 This is a comparison of brain tissue fluorescence imaging of experimental cerebral malaria model mice after injection of cGAMP or saline provided in Example 1 of the present invention;

[0021] Figure 7 This is a comparison chart of infection rates in experimental cerebral malaria model mice after injection of ASA-404 or normal saline provided in Example 1 of the present invention;

[0022] Figure 8 This is a comparison chart of the survival rates of the experimental cerebral malaria model mice injected with ODN 1826sodium combined with cGAMP or injected with normal saline provided in Example 2 of the present invention;

[0023] Fig. 9 This is a comparison chart of infection rates in the experimental cerebral malaria model mice provided in Example 2 of the present invention after injection of ODN 1826sodium combined with cGAMP or injection of normal saline. DETAILED DESCRIPTION

[0024] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] cGAMP disodium (Synonyms: Cyclic GMP-AMP disodium; 3', 3'-cGAMP disodium) was purchased from MCE Company with catalog number HY-110385 and a purity of 99.34%.

[0027] ODN 1826 sodium (Synonyms: CpG 1826 sodium) was purchased from MCE Company with the catalog number HY-146245C and a purity of 98.79%.

[0028] Vadimezan (Synonyms: DMXAA; ASA404) was purchased from Selleck Company with catalog number S1537 and a purity of 99.94%.

[0029] 1. Experimental model construction:

[0030] The role of cGAMP in preventing cerebral malaria was studied using the mouse experimental cerebral malaria model, an animal model of human cerebral malaria. Female C57BL / 6 mice aged 6 to 8 weeks were used, and each C57BL / 6 mouse was intraperitoneally injected with 1*10 5 The ANKA strain of Plasmodium berghei (PbA) was used to infect mice with malaria (cerebral malaria usually develops 5 to 9 days after infection, and mice with cerebral malaria will die rapidly within 1 day after the onset of typical cerebral malaria symptoms).

[0031] 2. Drug trials:

[0032] 2.1. Verification of the effect of cGAMP injection on the first day

[0033] Experimental cerebral malaria model mice were divided into two groups. One group was injected with 20 μg cGAMP (diluted to 200 ul with saline) through the tail vein on the first, third and fifth days after infection. The other group was injected with 200 ul saline through the tail vein on the first, third and fifth days after infection as a control. The survival rate was calculated and the infection rate was calculated by Giemsa staining.

[0034] Depend on Figure 1 The results show that PbA are mice infected with the ANKA strain of Plasmodium berghei, and PbA+cGAMP are the mice in the drug-treated group. The mice in the PbA group began to die on the 6th day of infection with Plasmodium berghei due to nerve damage caused by cerebral malaria, and all died on the 10th day; the mortality rate of the mice in the drug-treated group decreased after being injected with cGAMP, and the survival rate dropped to more than 80% only on the 8th-11th day. It can be seen that the survival rate of mice injected with cGAMP was greatly improved.

[0035] Depend on Figure 2 The results show that the infection rate was determined by Giemsa staining, using the ratio of the number of infected worms to the total number of cells. The results showed that the infection rate of the mice in the drug-treated group was slightly lower than that of the mice in the WT group.

[0036] 2.2 Validation of the non-experimental cerebral malaria model

[0037] Male C57BL / 6 mice aged 6 to 8 weeks were used, and each C57BL / 6 mouse was intraperitoneally injected with 1*10 5 Plasmodium yoelii YM was introduced to infect mice with malaria, and the infected mice were evenly divided into two groups. One group was injected with 20 μg cGAMP (diluted to 200 ul with saline) through the tail vein on the first, third, and fifth days after infection, and the other group was injected with 200 ul saline through the tail vein on the first, third, and fifth days after infection as a control, and the survival rate was calculated.

[0038] Depend on Figure 3 The results show that in the non-experimental cerebral malaria model, C57BL / 6 mice infected with Plasmodium yoelii YM strain, cGAMP does not play a role in prolonging survival time.

[0039] 2.3. Verification of cGAMP onset time

[0040] In the experimental cerebral malaria model, 200ul of normal saline was injected into the tail vein of each mouse in the control group (WT group), and 20μg of cGAMP (diluted to 200ul with normal saline) was injected into the tail vein of each mouse in the experimental groups (1D, 3D, 5D, 5D+6D groups) on the 1st, 3rd, and 5th days after infection, and 20μg of cGAMP (diluted to 200ul with normal saline) was injected into the tail vein continuously on the 5th and 6th days. The survival rate of each group was calculated.

[0041] Depend on Figure 4 The results showed that only the single drug administration on the 3rd day achieved a significant extension of survival, while the results of the other experimental groups only extended the survival time by 5 days at most compared with the control group, and all of them died in the end. Therefore, the results showed that cGAMP played a certain protective role in the early stage.

[0042] 2.4 In vivo mouse imaging experiment

[0043] C57BL / 6 mice were taken and each C57BL / 6 mouse was intraperitoneally injected with 1*10 5 The ANKA strain of Plasmodium berghei was tagged with Luc (luciferase reporter gene). Luciferase was injected intraperitoneally according to the weight of mice to make the parasites glow. The mice were divided into two groups. One group was injected with 20μg cGAMP (diluted to 200ul with saline) through the tail vein on the first, third and fifth days after infection. The other group was injected with 200ul saline through the tail vein on the first, third and fifth days after infection as a control. The fluorescence intensity was counted on the seventh day. After the statistics, the mouse brain tissue was taken out for imaging.

[0044] Depend on Figure 5 The results of A, B, and C showed that the fluorescence intensity of mice in the drug-treated group was significantly lower than that in the WT group, proving that cGAMP can effectively inhibit the development of the ANKA strain of Plasmodium berghei in mice.

[0045] Depend on Figure 6 The results showed that the number of parasites in the brain tissue of mice in the drug-treated group was significantly less than that in the WT group, indicating that the risk of cerebral malaria in mice was significantly reduced after drug administration.

[0046] 2.5 Non-CDNs agonist control

[0047] In the experimental cerebral malaria model, 200ul of normal saline was injected into the tail vein of the control group (WT group), and 500ug of ASA-404 (diluted to 200ul with normal saline) was injected intraperitoneally on the 1st, 3rd, and 5th days of infection, and the infection rate was counted.

[0048] Depend on Figure 7 The results showed that the non-CDNs agonist ASA-404 did not have a significant effect in preventing cerebral malaria.

[0049] Example 2

[0050] 1. Experimental model construction:

[0051] The role of cGAMP in preventing cerebral malaria was studied using the mouse experimental cerebral malaria model, an animal model of human cerebral malaria. Six to eight-week-old C57BL / 6 mice were used and each C57BL / 6 mouse was intraperitoneally injected with 1*10 5 The ANKA strain of Plasmodium berghei (PbA) was used to infect mice with malaria (cerebral malaria usually develops 5 to 9 days after infection, and mice with cerebral malaria will die rapidly within 1 day after the onset of typical cerebral malaria symptoms).

[0052] 2. Experimental methods

[0053] Experimental cerebral malaria model mice were divided into two groups. In one group, each mouse was injected with 20 μg cGAMP (diluted to 200 ul with saline) through the tail vein and 50 ug ODN 1826sodium (diluted to 200 ul with saline) through the peritoneal cavity on the first, third and fifth days after infection. In the other group, each mouse was injected with 200 ul saline through the tail vein and 200 ul saline through the peritoneal cavity on the first, third and fifth days as a control. The survival rate and the infection rate were calculated by Giemsa staining.

[0054] Depend on Figure 8 , 9 The results show that the compound ODN 1826sodium, a TLR9 agonist, was used in combination with cGAMP to prolong the survival time of mice and reduce the parasite infection rate in mice. It can not only prevent cerebral malaria but also reduce the amount of parasites in the body.

[0055] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Application of cGAMP as a STING agonist in the preparation of drugs for the prevention of cerebral malaria.

2. The use according to claim 1, characterized in that: The cGAMP is 3'3'-cGAMP.

3. The use according to claim 1, characterized in that: The prevention of cerebral malaria is to reduce the mortality rate of cerebral hemorrhage caused by Plasmodium infection.

4. The use according to claim 1, characterized in that: cGAMP was diluted with saline and injected into the tail vein of infected mice.

5. The application of cGAMP combined with TLR9 agonist in the preparation of drugs for preventing cerebral malaria.

6. The use according to claim 5, characterized in that: The TLR9 agonist is ODN 1826sodium.

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