CN106265715B

CN106265715B - Application of cyclic dinucleotide cGAMP in preparation of medicine for preventing senile dementia

Info

Publication number: CN106265715B
Application number: CN201510252849.2A
Authority: CN
Inventors: 张跃茹; 向道凤
Original assignee: Hangzhou Star Bioscience Co Ltd
Current assignee: Hangzhou star bioscience Co., Ltd.
Priority date: 2015-05-19
Filing date: 2015-05-19
Publication date: 2020-02-07
Anticipated expiration: 2035-05-19
Also published as: CN106265715A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of cyclic dinucleotide cGAMP in preparation of health care products. The research of the invention shows that the cyclic dinucleotide cGAMP is used as an activator of a human body natural immune signal pathway and a reinforcing agent of a natural immune system, and achieves the immune defense effects of preventing various virus infections, inhibiting the occurrence and the development of tumors, preventing senile dementia and the like which are important for disease prevention and body building by strengthening the immune capacity of the natural immune system. Animal acute toxicity experiments show that the cGAMP has no obvious acute toxicity, so that the cGAMP has potential application of preventing virus infection and resisting tumor as an endogenous second messenger capable of regulating natural immune response. cGAMP can be used as additive for preparing health product or special diet.

Description

Application of cyclic dinucleotide cGAMP in preparation of medicine for preventing senile dementia

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of cyclic dinucleotide (cGAMP) in preparation of a medicine for preventing senile dementia.

Background

The immune system of human body can recognize and eliminate non-self antigen and play the role of immune protection including anti-infection immunity, tumor immunity, etc. The human immune system can prevent and eliminate pathogen invasion, neutralize toxic metabolite or other foreign matter invasion. When immune surveillance is inadequate, it can lead to the development of tumors and persistent infection by the virus.

The free DNA present in the cytoplasm is always taken as a potential danger signal by the host's innate immune system, but the mechanisms by which the immune system recognizes and clears these danger signals are not well defined. In recent years, it has been found that a DNA receptor (DNAsensor) is a bridge for host to sense DNA and immune defense, and more than 10 DNA receptors have been discovered at present, while an interferon stimulating gene (STING), which is a key linker molecule in the downstream of DNA sensing pathway, plays an important role in sensing cytoplasmic DNA and immune defense signaling, and DNA in cytoplasm can activate STING through DNA receptor to produce type I interferon and other cytokines, thereby initiating immune response of the body. In recent years, there has been significant progress in the mechanistic study of how free DNA in the cytoplasm activates STING, which in turn initiates an immune response in vivo to produce an antiviral or antibacterial effect. Recently, a novel nucleic acid transferase, cGAS (cyclicGMP-ampsynthsase), which recognizes DNA and produces an endogenous cyclic dinucleotide, cGAMP (cyclicGMP-AMP), which is an activator of STING, has been found in mammalian cells. A recent study on HIV infection strongly suggests how STING senses the HIV-infected body via cGAS. In the human monocyte line, THP-1, HIV infection activates cytosolic cGAS and through it synthesizes endogenous cGAMP, activates the linker protein STING, which controls the viral infectious disease by recruiting TBK-1, phosphorylating IRF-3, and entry of activated IRF-3 into the nucleus to promote transcription of the interferon gene, producing IFN-thrin and CXCL 10. Furthermore, cGAS knock-outs in mouse (L929) or human cell lines (myeloid-derived macrophages, i.e., MDM and dendritic cells) would block the interferon response induced by the body's immune system against HIV, murine leukemia virus or simian immunodeficiency virus, and therefore these studies demonstrate that the cGAS-STING pathway is an important sensing pathway for viral infection in vitro (XinLiandPingweili, et., Immunity, 2013, 39(6), 1019-.

The expression of the receptor-regulated signal transduction has been shown to trigger conformational changes in STING, leading to recruitment and activation of the protein kinase TBK1 in the signal complex, the transcription factor IRF3 also subsequently enters the signal complex and is phosphorylated by TBK1, the phosphorylated IRF3 forms oligomers and is transported to the nucleus, initiating the expression of interferon β, interferon β is capable of regulating the expression of more than two hundred interferon-stimulated genes, which are capable of down-regulating protein synthesis, promoting cellular growth arrest and cell growth arrest, thus leading to the formation of a unique anti-viral immune response, which is an essential anti-viral response to cGAS, and which is likely to be used in the induction of a innate anti-viral immune response in mice.

Disclosure of Invention

The invention aims to provide application of cGAMP in preparing anti-senile dementia drugs.

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are given to better illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1: preparation of cGAMP

cGAMP (cyclic-GMP-AMP) is synthesized catalytically by cyclic cGMP-AMP dinucleotide synthetase (cGAS) under activation conditions after binding dsDNA according to literature procedures. The purity is more than 98%. (PingweiLi, et., Immunity, 2013, 39(6), 1019-

Example 2: acute toxicity study of cGAMP

Experimental Material

20 ICR mice (purchased from Shanghaisleke laboratory animals, Limited liability company [ laboratory animal quality certification number: SCXK (Shanghai) 2007-0005]), each half of male and female, the weight of the mice is 18-22 g, and the mice are fed with pellet feed and can freely eat and drink water. cGAMP was prepared from example 1 and formulated with physiological saline into a solution at a concentration of 200 mg/mL.

Experimental methods

ICR mice were injected with 5mg/kg cGAMP by single tail vein injection at body weight, and mice were observed for toxicity and death within 14 days after administration. As a result, it was found that the mice were normally active after a single tail vein injection. Mice did not die within 14 days after intravenous cGAMP injection, and on day 15, all mice were sacrificed, dissected, and examined for each organ, with no obvious lesions.

Results of the experiment

The result of the acute toxicity experiment shows that the maximum MTD (maximum tolerated dose) of the intravenous cGAMP is not less than 5g/Kg, which indicates that the cGAMP has no obvious acute toxicity.

Example 3: cGAMP with tumor immunoprophylaxis effect

Experimental Material

cGAMP was prepared from example 1 and formulated with physiological saline into a solution at a concentration of 200 mg/mL. 20 ICR mice (purchased from Shanghaisleke laboratory animals Limited liability company [ laboratory animal quality certification number: SCXK (Shanghai) 2007-0005]), male animals with weight of 18-22 g were fed with pellet feed, and had free food intake and drinking. Tumor cell lines: human gastric cancer cell line MNK-45, human lung adenocarcinoma cell line A549, human colon cancer cell line Lovo, human hepatoma cell line SMMC-7721, human prostate cancer cell line PC-3 and human pancreatic cancer cell SW1990, all purchased from cell banks of Chinese academy of sciences.

Experimental methods

Negative control, physiological saline solution; positive control, 5-fluorouracil (5-FU) anticancer agent (dose 5 mg/kg). cGAMP was administered intraperitoneally (5mg/kg dose) for 7 days, 1 injection per day, with 8 animals per group. Cancer cells were cultured, passaged, and cells were collected at the logarithmic phase of the cells to prepare a cell suspension with a concentration of 1.0X 106/ml. Mice were injected with cGAMP for 7 consecutive days, followed by axillary injection of 50 microliters of cancer cells suspension in the right forelimb. The negative control group (i.e., the group injected with saline solution), the positive control group (i.e., the group injected with 5-FU), and the cGAMP group (i.e., the group injected with cGAMP) were injected 1 time a day, and were continuously injected for 14 days, and the antitumor immune defense effect of cGAMP was observed.

The experimental results are as follows: mice injected with cGAMP were able to efficiently immune-defend against tumor development, and tumors appeared much later than the control group for 3-5 days, compared to mice not injected with cGAMP. Moreover, 5-FU and cGAMP can obviously inhibit the growth of tumors, and the weight average of the tumors after 14 days of cGAMP injection is obviously lower than that of a negative control group, which indicates that the cGAMP has the functions of immunoprophylaxis and antitumor.

Example 4: cGAMP with effect of preventing senile dementia

Experimental Material

cGAMP was prepared from example 1 and formulated with physiological saline into a solution at a concentration of 200 mg/mL. ICR mice (purchased from Shanghaisleke laboratory animals, LLC [ laboratory animal quality qualification number: SCXK (Shanghai) 2007-0005]) weighing 20-25g, were fed with pelleted feed, and were allowed to ingest food and drink water freely.

Experimental methods

(1) And 20 healthy male mice with the weight of 20-25g are divided into a group (10) with ice water swimming (CWS) and a group (10) with ice water swimming. The CWS group swims in ice water for 5 minutes. The CWS group was sacrificed 5 after 5 minutes on ice water swimming and 5 more after 12 hours. Control mice were sacrificed 12 hours after 5 minutes of ice water swimming. After the mice are sacrificed, hippocampus is taken out, protein is extracted, and the content of phosphorylated Tau protein is detected by WesternBlot.

(2) 5 healthy male mice, weighing 20-25g, were divided into groups of daily cGAMP (5mg/Kg), CWS + cGAMP (5). The control group, i.e., the CWS group (5) was injected with physiological saline, and after the intraperitoneal injection of cGAMP for 7 days, mice in the CWS + cGAMP group and the CWS group were allowed to swim in ice water for 5 minutes, all the mice were sacrificed 12 hours after the swimming for 5 minutes to take hippocampus, and protein was extracted, and Westernblot was performed to detect the amount of phosphorylated Tau protein in the hippocampus.

And (4) experimental conclusion:

(1) the content of phosphorylated Tau protein in the hippocampus of the normal group of mice is obviously lower than that of the ice water swimming group, which proves that the ice water swimming can cause the brain emergency reaction of the mice, so that the content of phosphorylated Tau protein in the hippocampus is obviously increased, and the hyperphosphorylation and aggregation of Tau protein is an important pathological characteristic of senile dementia.

(2) In the mice swimming in ice water, the content of phosphorylated Tau protein was significantly reduced in the cGAMP-injected group as compared with the non-injected group, and therefore, it was considered that cGAMP had a significant effect of reducing the increase in phosphorylated Tau protein content caused by ice water swimming stress. The results show that the cGAMP has the function of enhancing immune defense and has the potential effect of preventing the development of senile dementia.

Claims

The application of cGAMP in preparing medicine for preventing senile dementia.