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

Abstract

The invention discloses a medical application of Nobilitin in preparation of an anti-neospora small molecule drug, and provides a novel medical application of Nobilitin, and the treatment effect of the drug on neospora is proved in two aspects of acting on host cells and mouse animal experiments. According to the invention, a new candidate drug Nabiltin is provided for the treatment of neospora caninum, and a foundation is laid for searching a novel drug for resisting neospora caninum disease in the future.

Description

Technical Field

[0001] This invention discloses the medical use of Nobiletin in the preparation of anti-neosporidioid small molecule drugs, providing a new medical use for Nobiletin in the preparation of drugs for treating neosporidiosis, in the field of antiparasitic drug technology. Background Technology

[0002] Neosporosis is a disease caused by Neosporidia of the phylum Apicocomplexa (Neosporidia). Neospora caninum Parasitic diseases caused by these parasites are widely distributed globally and cause serious economic losses to the aquaculture industry.

[0003] Neosporidia can infect a variety of mammals, causing damage to their reproductive and nervous systems, primarily leading to abortion in females and neonatal death in animals such as cattle and sheep. Currently, there is no effective vaccine or specific treatment for this disease; therefore, the development of novel antiparasitic drugs is crucial. Summary of the Invention

[0004] This invention discloses the medical use of Nobiletin in the preparation of anti-neosporidioid small molecule drugs, which has significant medical effects and can be used to prepare drugs for treating neosporidiosis.

[0005] The Nobiletin involved in this invention has a molecular weight of 402.39 and a chemical formula of C. 21 H 22 O8 has the following stability: it can be stored for 3 years in powder form at -20℃ and for 1 year in solution form (dissolved in DMSO) at -80℃. Its chemical structure is shown below:

[0006] The Nobiletin described in this invention can be formulated into an anti-neosporidis drug to inhibit the infection and proliferation of neosporidia, thereby achieving the effect of treating neosporidiasis.

[0007] This invention has found that activating the NLRP3 inflammasome and inducing pyroptosis can inhibit *Neospora* to some extent. The use of ROS inducers (such as gallic acid and ivermectin) and the pyroptosis inducer niclosamide can both exert anti-insect effects by inducing pyroptosis. There is potential to find novel anti-insect drugs by inducing pyroptosis. Nobiletin can induce apoptosis and pyroptosis in breast cancer cells through the miR-200b / JAZF1 / NF-κB axis; therefore, this invention starts with this drug and discovers a new anti-*Neospora* drug.

[0008] This invention, based on the mechanism by which activation of the NLRP3 inflammasome is beneficial for combating Neosporidia, seeks drugs that can synergistically activate host cell pyroptosis to inhibit Neosporidia infection. After activating the NLRP3 pathway with this drug, the proliferation of Neosporidia within cells is detected. Subsequently, animal experiments are conducted in Vero cell models (cell invasion and infection experiments) and mouse models to observe its therapeutic effects on Neosporidia invasion of host cells, proliferation within host cells, and parasitic infection in mouse brain, heart, and lung tissues. Furthermore, this invention provides the working concentration and potential pathway of action of this inducer to exert its anti-Neosporidiotic effect, providing a new target for anti-Neosporidiotic infection and a new candidate drug for future treatment of Neosporidia. The drug can be formulated into any dosage form containing the active ingredient Nobiletin.

[0009] The mechanism of action of the anti-Neosporidis drug of this invention includes the following: First, the level of IL-1β produced by the host in response to Neosporidia stimulation is detected using an ELISA kit to analyze whether the drug synergistically induces pyroptosis in host cells. Second, the proliferation of Neosporidia in Vero cells is detected using Giemsa staining and qPCR. Finally, a mouse infection model is established, and the drug's ability to combat Neosporidia infection is evaluated by detecting mouse survival, pathological changes in brain, heart, and lung tissues, and the number of parasites.

[0010] The positive effects of this invention are: it provides a new medical application for Nobiletin, demonstrating its significant inhibitory effect on Neosporidia infection. The therapeutic efficacy of this drug against Neosporidia has been confirmed through both host cell and mouse animal experiments. This invention provides Nobiletin as a new candidate drug for the treatment of Neosporidia, laying the foundation for the future search for novel drugs against Neosporidia. Attached Figure Description

[0011] Figure 1 This invention validates the activation of mouse peritoneal macrophages by Nobiletin. Figure 2 This invention demonstrates the inhibitory effect of Nobiletin on Neosporidium proliferation in macrophages. Figure 3 This invention demonstrates the inhibitory effect of Nobiletin on the proliferation of Neosporidia in Vero cells. Figure 4 This invention demonstrates the inhibitory effect of Nobiletin on acute infection of Neosporidium in mice. Figure 5 This invention demonstrates the protective effect of Nobiletin against the survival of mice infected with Neosporidium. Figure 6This invention demonstrates the inhibitory effect of Nobiletin on Neosporidium infection in sheep. Detailed Implementation

[0012] The following describes specific embodiments of the present invention. However, those skilled in the art should understand that these are merely illustrative examples. 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 embodiments without departing from the principles and essence of the present invention, and all such changes and modifications fall within the scope of protection of the present invention.

[0013] Example 1

[0014] The pathway of Nobiletin acting on primary mouse peritoneal macrophages: After activating the NLRP3 pathway in host cells with Nobiletin, the effect of Nobiletin on the infection of host cells by Neospora was observed, and the experimental data were analyzed by biostatistics.

[0015] Macrophages were infected with Neosporidium tachyzoites (tachyzoites:cells = 1:1) for 3 h, followed by incubation with Nobiletin (25 µM and 50 µM) for another 20 h. Results showed that compared with the Neosporidium-only treatment group, the IL-1β levels in the cell culture supernatant increased by 34.0% and 23.6%, respectively. P <0.01 and P <0.05) (as attached) Figure 1 A macrophage infection model was treated with Nobiletin (10 µM, 25 µM, and 50 µM). Cell pellets were collected, DNA was extracted, and qPCR was used to detect the number of neosporidia in the cells. The results showed that 10 µM, 25 µM, and 50 µM Nobiletin reduced the number of intracellular neosporidia by 47.5%, 63.6%, and 76.6%, respectively. P <0.05、 P <0.01 and P <0.01). Analysis of cell infection rate and the number of tachyzoites within nauplii vacuoles after Giemsa staining revealed that Nobiletin treatment significantly reduced the Neospora cell infection rate and the number of tachyzoites within nauplii vacuoles (see attached). Figure 2 ).

[0016] Example 2

[0017] 1. The blocking effect of Nobiletin on Neosporidium invasion of Vero cells and its inhibitory effect on intracellular proliferation. This invention uses the pyroptosis inducer Nobiletin as an example to demonstrate its blocking and inhibitory effects on Neosporidium invasion of Vero cells and intracellular proliferation, and performs biostatistical analysis on the experimental data.

[0018] After infecting Vero cells with Neosporidium tachyzoites (tachyzoite:cell = 1:1) for 3 h, Nobiletin (10 µM, 15 µM, 20 µM) was added to the culture medium and incubated for another 24 h. Results showed that after 24 h, qPCR analysis of the number of Neosporidium-bearing cells revealed that 10 µM, 15 µM, and 20 µM Nobiletin reduced the number of intracellular Neosporidium by 54.6%, 58.53%, and 64.86%, respectively. P <0.001). Analysis of cell infection rate and the number of tachyzoites within the vacuoles of *Neospora* after Giemsa staining revealed that 10 µM, 15 µM, and 20 µM of Nobiletin reduced the *Neospora* cell infection rate by 37.2%, 37.4%, and 39.8%, respectively, and also significantly reduced the number of tachyzoites within the vacuoles. P <0.001) (as attached) Figure 3 ).

[0019] 2. The therapeutic effect of Nobiletin on Neosporidium infection in mice This invention uses the pyroptosis activator Nobiletin as an example to observe its therapeutic effect on a mouse model infected with Neosporidium.

[0020] Intraperitoneal inoculation of mice 2×10 7 An acute infection model of *Neospora* was established in mice using tachyzoites. Three days after infection, mice were administered nobiletin or a solvent via gavage daily for three consecutive days. Groups were established including a PBS group, a 75 mg / kg administration group, a simple tachyzoite challenge group, a tachyzoite challenge + 25 mg / kg administration group, a tachyzoite challenge + 50 mg / kg administration group, and a tachyzoite challenge + 75 mg / kg administration group. Changes in mouse body weight and mortality were observed. After administration, mice were sacrificed, and their peritoneal cavities were flushed with pre-cooled PBS solution. The flushing fluid was collected by centrifugation. Brain, heart, and lung tissues were ground and genomic DNA was extracted. The number of *Neospora* tachyzoites per 200 ng of DNA was detected by qPCR using Nc-5 specific primers. The primer sequences are as follows: Upstream: 5′-TCCCTCGGTTCACCCGTTCACACAC-3′; Downstream: 5′-CACGTATCCCACCTCTCACCGCTACCA-3′; The results showed that the amount of *Neospora* worms in the peritoneal lavage fluid of mice decreased by 79.45%, 92.58%, and 86.99% compared to the *Neospora* group in the 25 mg / kg Nob, 50 mg / kg Nob, and 75 mg / kg Nob groups, respectively. The amount of *Neospora* worms in the heart decreased by 92.97%, 96.51%, and 87.56% compared to the *Neospora* group in the 25 mg / kg Nob, 50 mg / kg Nob, and 75 mg / kg Nob groups, respectively. The amount of *Neospora* worms in brain tissue decreased by 51.54%, 68.19%, and 71.29% compared to the *Neospora* group in the 25 mg / kg Nob, 50 mg / kg Nob, and 75 mg / kg Nob groups, respectively. The number of neosporidia in the lungs was measured. Compared with the *Neospora* group, the 25 mg / kg Nob group, 50 mg / kg Nob group, and 75 mg / kg Nob group showed a decrease in neosporidia number of 82.50%, 87.67%, and 74.74%, respectively. This indicates that a Nobiletin concentration of 50 mg / kg can significantly reduce the number of neosporidia in various tissues of a mouse model infected with *Neospora*. Comparing body weight among the groups, the Nobiletin-treated group showed faster weight recovery when the percentage of body weight decreased to its lowest point on day 3 (see attached figure). Figure 4 ).

[0021] Intraperitoneal inoculation of mice 4×10 7 Survival experiments were conducted on tachyzoites of *Neospora*. Three days after infection, mice were administered nobiletin or a solvent via gavage daily for one week. Groups were established including a PBS group, a 75 mg / kg administration group, a simple infection group, an infection + 25 mg / kg administration group, an infection + 50 mg / kg administration group, and an infection + 75 mg / kg administration group. Changes in mouse body weight and mortality were observed.

[0022] The results showed that compared with the infection group, the treatment group recovered body weight faster and had a higher survival rate. The survival rate increased to 60% in the group treated with worm challenge plus 50 mg / kg, and to 40% in the group treated with worm challenge plus 25 mg / kg (see attached). Figure 5 ).

[0023] 3. The therapeutic effect of Nobiletin on Neosporidia infection in sheep This invention uses the pyroptosis activator Nobiletin as an example to observe its therapeutic effect on a sheep model infected with Neosporidium.

[0024] Sheep infection model: intravenous inoculation of sheep 1×10 9One week after infection with *Neospora* tachyzoites, sheep were administered nobiletin or a solvent via gavage daily for 7 consecutive days. Separate groups were established: a PBS group, a simple infection group, and an infection + 30 mg / kg drug group. Sheep weight and rectal temperature were measured. On day 45 of infection, anesthetized sheep were euthanized by venous bleeding. Heart, liver, spleen, lung, kidney, and brain tissues were collected from each group. The tissues were minced and mixed, and soybean-sized portions were ground and genomic DNA extracted. The number of *Neospora* tachyzoites per 500 ng of DNA was detected by qPCR (primer sequences as above). The results showed that, compared with the Neosporidium group, the Nobiletin-treated group had a reduced number of worms in all tissues, with a 70.4% reduction in the number of worms in the heart, a 28.6% reduction in the number of worms in the liver, a 72.6% reduction in the number of worms in the spleen, and an 87.1% reduction in the number of worms in the lungs, with significant differences. P <0.05); the amount of worms in kidney tissue decreased by 71.6%; the amount of worms in brain tissue decreased by 80.9% (see attached). Figure 6 ).

[0025] Based on the above experimental results, the anti-neosporidis drug of the present invention, through two pathways—acting on host cells and infecting mouse animal models—significantly inhibits the invasion of neosporidia into host cells, the proliferation of parasites within host cells, and the number of parasites in various tissues of mice. This indicates that Nobiletin can exert a good anti-neosporidis effect and can be used as a novel candidate drug for the treatment of neosporidiasis.

Claims

1. Medical applications of Nobiletin in the preparation of small molecule drugs against Neosporidia; The aforementioned Nobiletin has a molecular weight of 402.39 and a chemical formula of C. 21 H 22 O8, the chemical structural formula of which is shown below: 。 2. To formulate a drug in any dosage form containing the active ingredient Nobiletin.

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