Medical application of Niclosamide in preparation of anti-neosporidium micromolecular medicine

Abstract

The invention discloses a medical application of niclosamide in preparation of an anti-neospora micromolecule drug, provides a novel medical application of niclosamide, and discovers a chemical drug capable of remarkably inhibiting neospora cell infection and mouse model brain, heart and lung tissue infection. The treatment effect of the compound on neospora caninum is proved through two aspects of animal experiments acting on host cells and mouse models, and the compound can be used for preparing novel anti-neospora caninum drugs.

Description

Technical Field

[0001] The invention discloses a new medical use of a compound niclosamide, which is used for preparing a novel anti-neosporidial drug and has an obvious therapeutic effect on neosporosis, belonging to the technical field of anti-parasitic drugs. Background Art

[0002] Neosporosis is a disease caused by the protozoan Neosporosis ( Neospora caninum ). Neospora has a wide host range, and its infection can cause miscarriage and nervous system damage in animals, especially in cattle, where it shows extremely high pathogenicity. It has caused serious economic losses to the global animal husbandry, especially to dairy and beef cattle production. Currently, there are limited treatments for Neospora infection. Although there are some antiparasitic drugs on the market, they are often unable to completely eliminate parasites in the body, leading to chronic and recurrent infections. There is still a lack of specific therapeutic drugs for Neospora. Summary of the invention

[0003] The present invention provides a medical use of Niclosamide in the preparation of an anti-neosporosis small molecule drug, which is used for inhibiting the infection and proliferation of neosporosis to achieve the effect of treating neosporosis.

[0004] The compound niclosamide of the present invention has the following chemical structure:

[0005] Its chemical formula is C 13 H 8 C l2 N 2 O 4 , molecular weight is 327.12.

[0006] The content of the anti-neospora drug of the present invention includes: according to the mechanism that the activation of NLRP3 inflammasome is beneficial to anti-neospora, a drug that can cooperate with neospora to activate the second signaling pathway of the host is found, and after the drug is used to activate the second signaling pathway of NLRP3, the proliferation of neospora in cells is detected. Then, through cell invasion and infection experiments on Vero cell models and animal experiments on mouse models, the therapeutic effect of the drug on the invasion of neospora into host cells, proliferation in host cells and parasite infection in mouse brain, heart and lung tissues is observed, and the working concentration and potential action pathway of the drug to exert anti-neospora effect are provided, so as to provide a new action target for anti-neospora infection and provide a new candidate drug for the future treatment of neosporosis.

[0007] The action pathway of the anti-neosporidial drug of the present invention includes the following contents: First, niclosamide was used to assist Neospora in stimulating the host to produce LDH and IL-1β by LDH and ELISA. The activation effect of the drug on the host cell NLRP3 signaling pathway was analyzed, and the invasion of Neospora into host cells after the activation of the host cell NLRP3 pathway was evaluated; secondly, the proliferation of Neospora in Vero host cells was detected by Giemsa staining and qPCR. The number of parasites in the brain, heart, and lung tissues of mice acutely infected with Neospora was detected by qPCR. The ultrastructural damage caused by the drug to Neospora tachyzoites was observed by transmission electron microscopy. The effect of the drug on the mitochondria of Neospora tachyzoites was detected by JC-1 staining and ATP detection kit.

[0008] NLRP3 inflammasomes participate in the regulation of host resistance to Neospora, blocking Neospora infection and clearing infected Neospora from the host side. Activation of NLRP3 inflammasomes usually requires two signals, pathogen-associated molecular patterns or damage-associated molecular patterns as the first signal pathway, and the second signal usually involves mitochondrial damage, potassium efflux, calcium influx or ROS production. The second signal pathway of the present invention is a target, providing a new type of drug for anti-Neospora infection, and providing a new idea for the treatment of Neosporosis.

[0009] The positive effects of the present invention are: providing a new medical use of niclosamide, discovering a chemical drug that can significantly inhibit Neospora cell infection and brain, heart and lung tissue infection in a mouse model, confirming its therapeutic effect on Neospora through animal experiments on host cells and mouse models, and can be used to prepare a new anti-Neospora drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The niclosamide of the present invention partially controls the proliferation of Neospora via NLRP3-assisted macrophages; Figure 2 The blocking effect of niclosamide of the present invention on the invasion and proliferation of Neospora; Figure 3 The blocking effect of niclosamide of the present invention on Neospora infection in mice; Figure 4 The effect of niclosamide of the present invention on the ultrastructure of tachyzoites of Neospora; Figure 5 The invention discloses the influence of niclosamide on mitochondria of neospora tachyzoites. DETAILED DESCRIPTION

[0011] The following describes the specific implementation methods of the present invention, but those skilled in the art should understand that this is merely an example, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these implementation methods without departing from the principle and essence of the present invention, and these changes and modifications all fall within the scope of protection of the present invention.

[0012] Example 1 The present invention takes niclosamide, an activator of the second signaling pathway of NLRP3, as an example to demonstrate its therapeutic effect on the invasion of Neospora cells and intracellular proliferation.

[0013] The pathway of niclosamide's action on primary mouse peritoneal macrophages: After activating the host cell NLRP3 pathway with niclosamide, its effect on host cells infected with Neospora was observed, and the data obtained from the experiment were subjected to biostatistical analysis. After macrophages were infected with Neospora tachyzoites (tachyzoites: cells = 1:1) for 3 h, niclosamide (1 µM) was added to the culture medium and incubated with host cells for 24 h. The results showed that compared with the group treated with Neospora alone, at 24 h, the levels of LDH and IL-1β in the cell culture supernatant increased by 75.7% and 169.4%, respectively (P<0.01 and P<0.001); in order to verify whether niclosamide inhibits the proliferation of Neospora mainly through NLRP3, Nlrp3 - / - The results showed that the IL-1β level in WT and WT mouse macrophages was reduced by 83.2% (P<0.001), but Nlrp3 - / - In mouse macrophages, niclosamide's inhibitory effect on intracellular Neospora was only slightly affected. This suggests that niclosamide acts on host cells to partially activate the NLRP3 inflammasome to exert its anti-Neospora infection effect (such as attachment Figure 1 ).

[0014] Example 2

[0015] The present invention takes niclosamide, an activator of the second signaling pathway of NLRP3, as an example to demonstrate its therapeutic effect on the invasion of Neospora into Vero cells and intracellular proliferation, and performs biostatistical analysis on the data obtained from the experiment.

[0016] Vero cells were infected with Neospora tachyzoites (tachyzoites: cells = 1:1) for 3 h, and then niclosamide (0.25 µM and 0.5 µM) was added to the culture medium and incubated with the host cells for 24 h. The results showed that after 24 h, the load was detected by qPCR and it was found that 0.25 µM and 0.5 µM niclosamide reduced the number of intracellular Neospora by 82.2% and 89.4%, respectively (P<0.001). The cell infection rate and the number of tachyzoites in the parasite vacuole were analyzed by Giemsa staining. The results showed that 0.25 µM and 0.5 µM niclosamide reduced the infection rate of intracellular Neospora by 64.2% and 81.1%, respectively, and the number of tachyzoites in the parasite vacuole was also significantly reduced (P<0.001) (as shown in the attached Figure 2 ).

[0017] 2. The inhibitory effect of Niclosamide on tachyzoites of Neospora The present invention takes niclosamide, an activator of the second signaling pathway of NLRP3, as an example to demonstrate its inhibitory effect on tachyzoites of Neospora, and performs biostatistical analysis on the data obtained from the experiment.

[0018] Vero cells were infected with Neospora tachyzoites (tachyzoites: cells = 1:1) for 3 h, and then niclosamide (0.5 µM) was added to the culture medium and incubated with the host cells for 24 h. Transmission electron microscopy results showed that 0.5 µM niclosamide treatment caused a large number of vacuoles to appear inside Neospora tachyzoites, and the basic structure was severely damaged (such as the attached Figure 3 ).

[0019] Purified fresh Neospora tachyzoites were incubated with niclosamide (0.25 µM and 0.5 µM) for 2 h. The JC-1 staining results showed that in the DMSO-treated group, JC-1 mostly existed in the form of aggregates, while in the niclosamide-treated group, JC-1 gradually converted into monomers, and the higher the dose of niclosamide, the higher the monomer ratio. This indicates that niclosamide causes a decrease in the mitochondrial membrane potential of tachyzoites. Statistical analysis showed that 0.25 µM niclosamide caused an 87% decrease in the mitochondrial membrane potential of tachyzoites, and 0.5 µM niclosamide caused a 94% decrease in the mitochondrial membrane potential of tachyzoites (P<0.001). In addition, ATP content detection showed that the ATP content in tachyzoites treated with niclosamide decreased by 19.2% (P<0.05) (as shown in the attached figure). Figure 4 ).

[0020] 3. The therapeutic effect of Niclosamide on Neospora infection in mouse models The present invention takes niclosamide, an activator of the second signaling pathway of NLRP3, as an example to observe its therapeutic effect on a mouse infection model of Neospora.

[0021] Mice were intraperitoneally inoculated for 2 × 10 7 NC-1 tachyzoites were added to the mice. Three days after infection, mice were gavaged with niclosamide or solvent every day for one week. PBS group, pure worm group, 200 mg / kg group, worm+50 mg / kg group, worm+100 mg / kg group, worm+200 mg / kg group were set up to observe the weight changes and mortality of mice. After the end of the drug administration, the mice were killed, and their brain, heart, and lung tissues were ground and genomes were extracted. The number of Neospora tachyzoites in each 500 ng of DNA in each tissue was detected by qPCR (using Nc-5 specific primers, the primer sequences are: Upstream primer: 5′-TCCCTCGGTTCACCCGTTCACACAC-3′; Downstream primer: 5′-CACGTATCCCACCTCTCACCGCTACCA-3′; The results showed that after one week of administration of Niclosamide (50 mg / kg), the number of tachyzoites / 500ng DNA in the brain, heart, and lung of mice decreased by 52% (P<0.001), 6.3% (P>0.05), and 67.1% (P<0.05), respectively. After one week of administration of Niclosamide (100 mg / kg), the number of tachyzoites / 500ng DNA in the brain, heart, and lung of mice decreased by 70.7% (P<0.001), 58.3% (P<0.01), and 80.6% (P<0.01), respectively. After one week of administration of Niclosamide (200 mg / kg), the number of tachyzoites / 500ng DNA in the brain, heart, and lung of mice decreased by 79.8% (P<0.001), 50% (P<0.01), and 80.8% (P<0.01), respectively. This indicates that niclosamide at a concentration of 100 mg / kg can significantly reduce the number of parasites in various tissues of the mouse model infected with Neospora (such as attached Figure 5 ).

[0022] Based on the above experimental results, the anti-neosporidial drug of the present invention exerts significant inhibitory effects on the invasion of neospora into host cells, the proliferation of parasites in host cells, and the number of parasites in various tissues of mice by acting on host cells and infecting mouse animal models. This shows that niclosamide can play a good anti-neosporidial effect and can be used as a new candidate drug for the treatment of neosporosis.

Claims

1. The medical use of the compound Niclosamide in the preparation of small molecule drugs against Neospora; The chemical structure of the compound Niclosamide is as follows: ； Its chemical formula is C 13 H8C l2 N2O4, molecular weight is 327.

12.

2. The drug containing the chemical component Niclosamide can be made into any pharmaceutical dosage form in pharmacology.

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