Application of anemarrhenae b4 in preparing anti-malaria drugs

Abstract

This invention provides the application of Pulsatilla saponin B4 in the preparation of antimalarial drugs, which can significantly improve weight loss, peripheral blood parasitemia, and survival rate in mice infected with lethal Plasmodium. Its mechanism of action is not to directly kill Plasmodium, but to regulate the host immune system, including activating T / B cell immune responses, upregulating pro-inflammatory factors and interferon signaling pathways, and downregulating Arg1 expression. At the same time, Pulsatilla saponin B4 can alleviate immunopathological damage to organs such as the spleen and liver, restore the homeostasis of the spleen's immune microenvironment, and protect the normal structure of tissues. As a natural product, this compound has the characteristics of multiple targets and low toxicity, and has the dual advantages of anti-infection and immune homeostasis regulation, showing good prospects for clinical application and translation.

Description

Technical Field

[0001] This invention relates to the field of natural product application technology, and in particular to the application of Pulsatilla saponin B4 in the preparation of antimalarial drugs. Background Technology

[0002] Malaria remains a major parasitic disease posing a serious threat to human health. Its pathogenesis and outcome depend not only on the Plasmodium load but also on the strength and regulatory status of the host's immune response. Especially during the intraerythral phase of malaria, excessive or unbalanced immune inflammatory responses are significant causes of tissue damage, disease exacerbation, and insufficient immune protection. However, our understanding of the regulatory mechanisms of immunopathological damage during the intraerythral phase remains insufficient, and effective intervention strategies that balance anti-infection efforts with immune homeostasis are lacking.

[0003] Natural products have shown unique advantages in the prevention and treatment of infectious diseases due to their multiple targets, low toxicity and good immunomodulatory potential, but systematic research on their role in the immune regulation of malaria is still very limited.

[0004] In summary, the current understanding of the regulatory mechanisms of intraerythropathological damage is still insufficient, and there is a lack of effective intervention strategies that take into account both anti-infection and immune homeostasis. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an application of Pulsatilla saponin B4 in the preparation of antimalarial drugs, so as to overcome the shortcomings of the prior art.

[0006] This invention provides the application of Pulsatilla saponin B4 in the preparation of antimalarial drugs.

[0007] Furthermore, the antimalarial drug exerts its antimalarial effect by modulating the function of the host's immune system.

[0008] Furthermore, the antimalarial drug includes at least one of improving weight loss caused by malaria infection, peripheral blood parasitemia, improving infection survival rate, or reducing immunopathological damage caused by malaria infection.

[0009] Furthermore, the immunopathological damage includes at least one of the following: a)-g) a) Splenomegaly; b) Damage to the spleen's tissue structure; c) Increased erythrocyte adhesion in liver blood vessels; d) Increased white blood cell recruitment; e) Malaria pigment deposition; f) Damage to the thymus structure; g) Disruption of lymph node structure.

[0010] Furthermore, the antimalarial drug used to modulate the immune response of a host infected with malaria includes at least one of the following: a)-c) a) Promotes T cell and / or B cell activation; b) Regulate macrophage function; c) Promotes the activation of the interferon signaling pathway.

[0011] Furthermore, the promotion of T cell and / or B cell activation is manifested by increased mRNA expression levels of Cd3 and / or Cd19 in the spleen; Macrophage function is regulated by increased mRNA expression levels of Nos1, Tnf, and II6 and / or decreased mRNA expression levels of Arg1 in the spleen. The activation of the interferon signaling pathway is promoted by increased serum IFN-γ levels and / or increased mRNA expression levels of II12 and Cxcl9 in the spleen.

[0012] The pulsatilla saponin B4 disclosed in this invention, when used in the preparation of antimalarial drugs, has shown in experiments to significantly improve weight loss, peripheral blood parasitemia, and survival rate in mice infected with lethal Plasmodium falciparum. Its mechanism of action is not to directly kill Plasmodium falciparum, but rather to regulate the host immune system, including activating T / B cell immune responses, upregulating pro-inflammatory factors and interferon signaling pathways, and downregulating Arg1 expression. Simultaneously, pulsatilla saponin B4 can alleviate immunopathological damage to organs such as the spleen and liver, restore the splenic immune microenvironment homeostasis, and protect normal tissue structure. As a natural product, this compound possesses multi-target and low-toxicity characteristics, combining the dual advantages of anti-infection and immune homeostasis regulation, and has promising prospects for clinical application and translation. Attached Figure Description

[0013] Figure 1 The image shows the effect of Pulsatilla saponin B4 in the first embodiment of the present invention on improving the symptoms of P. yoelii 17XL model mice; Figure 2 This is a diagram showing the gene changes in the spleen of a wild-type mouse model infected with Pulsatilla saponin B4 in the second embodiment of the present invention. Figure 3 This is a diagram showing the pathological changes in the spleen, liver, thymus, and lymph nodes of infected wild-type mice caused by intervention with Pulsatilla saponin B4 in the third embodiment of the present invention. Figure 4 This is a diagram showing the multiple immunofluorescence staining of the spleen of an infected mouse in the third embodiment of the present invention.

[0014] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention.

[0015] The embodiments of the present invention will be further described below with reference to the accompanying drawings. Detailed Implementation

[0016] Female C57BL / 6 mice aged 6-8 weeks, weighing (20±2) g, were randomly divided into a model group and a Pulsatilla chinensis saponin B4 treatment group. Five dosage groups were established: 25, 40, 50, 60, and 100 mg / kg / day, with 15 mice in each group. All mice in the treatment groups were administered the corresponding dose of Pulsatilla chinensis saponin B4 by gavage daily for 7 days prior to infection, while the model group received an equal volume of the solvent. After pretreatment, all mice were intraperitoneally inoculated with 5×10⁵ Pulsatilla chinensis saponin B4. 6 Red blood cells infected with a lethal strain of Plasmodium johnsonii (Py17XL); Mouse body weight, peripheral blood parasitemia, and survival rate were monitored daily after infection. Results showed that, compared with the model group, the weight loss in all treatment groups was alleviated to varying degrees, with the 50, 60, and 100 mg / kg / day groups showing the most significant effects. Figure 1 A); Peripheral blood parasitemia levels were significantly reduced, and in a dose-dependent manner ( Figure 1 B); Survival rate was significantly improved, with the survival rate of the 50 mg / kg / day group exceeding 80% ( Figure 1 C). In vitro fluorescence detection showed that Pulsatilla saponin B4 had no inhibitory effect on the direct growth of Plasmodium parasites. Figure 1 D). This example demonstrates that Pulsatilla saponin B4 can effectively improve the outcome of malaria infection, and its effect is unrelated to direct insecticidal activity.

[0017] C57BL / 6 mice were used and divided into a model group and a 50 mg / kg / day Pulsatilla saponin B4 treatment group, with 15 mice in each group. Mice were sacrificed on day 5 post-infection, and total RNA was extracted from spleen tissue. The expression of immune-related genes was detected by RT-qPCR. Serum was also collected, and IFN-γ levels were detected by ELISA. The results showed that, compared with the model group, the mRNA levels of Cd3 and Cd19 in the spleen of mice in the drug-treated group were significantly increased. Figure 2 A-2B indicates that T cell and B cell immune responses are activated; the mRNA levels of Nos1, Tnf, and II6 are increased, while the mRNA level of Arg1 is decreased. Figure 2 E-2I indicates macrophage polarization towards the M1 type; elevated mRNA levels of II12 and Cxcl9 ( Figure 2 K-2L, increased serum IFN-γ levels ( Figure 2 J), indicating activation of the interferon signaling pathway. However, the neutrophil marker genes Mpo and Ly6g showed no significant changes (J). Figure 2 (C-2D). This embodiment demonstrates that Pulsatilla saponin B4 remodels the antimalarial immune response by specifically regulating T / B cells, macrophages, and interferon signaling pathways.

[0018] C57BL / 6 mice were used, and a model group and a 50 mg / kg / day Pulsatilla saponin B4 treatment group were set up according to the method in Example 1, with 15 mice in each group. On the 5th day after infection, the mice were sacrificed, and the spleen, liver, thymus, and lymph node tissues were collected for H&E staining and multiplex immunofluorescence staining. H&E staining results showed that in the model group mice, the spleen was significantly enlarged, the boundary between the white and red pulp was blurred, and the germinal centers were destroyed; a large number of erythrocytes adhered, leukocytes recruited, and malarial pigment deposited were observed in the liver vessels; the boundary between the thymic cortex and medulla was unclear; and the lymph node structure was disordered. In the drug-treated group mice, the splenomegaly was alleviated, the boundary between the white and red pulp was clear, and the germinal centers were normal; the pathological changes in the liver were significantly reduced; and the thymus and lymph nodes were clearly and intact. Figure 3 As shown. Multiplex immunofluorescence staining revealed that in the spleen of mice treated with the drug, the distribution of CD3⁺ T cells and CD19⁺ B cells tended to be normal, F4 / 80⁺ macrophage infiltration was reduced, and Ly6G⁺ neutrophils showed no significant changes. Figure 4 As shown in the example, this embodiment demonstrates that Pulsatilla saponin B4 can effectively alleviate multi-organ immunopathological damage caused by malaria and restore the homeostasis of the spleen's immune microenvironment.

[0019] C57BL / 6 mice were used and divided into a model group and five treatment groups (25, 40, 50, 60, and 100 mg / kg / day) of Pulsatilla saponin B4, with 15 mice in each group. Pretreatment and infection were performed according to the method in Example 1. Mice were sacrificed on day 5 post-infection, and spleen tissue was collected. The expression levels of key immune genes such as Cd3, Cd19, Nos1, Arg1, and Ifng were detected by RT-qPCR, and the effects of each dosage group were compared. The results showed that, compared with the model group, the 25 mg / kg / day group had no significant immunomodulatory effect; the 40 mg / kg / day group began to show an upregulation trend of Cd3, Cd19, and Nos1 and a downregulation trend of Arg1; the 50, 60, and 100 mg / kg / day groups all showed significant immunomodulatory effects, and there were no statistically significant differences among the three groups. Considering both efficacy and cost-effectiveness, 50 mg / kg / day was the preferred dose. This example further verified the dose-dependent immunomodulatory effect of Pulsatilla saponin B4 and determined the effective dose range.

[0020] In summary, the above embodiments systematically verified the application value of Pulsatilla saponin B4 in the preparation of antimalarial drugs from the perspectives of pharmacodynamics, immune regulation mechanism, pathological improvement, and dosage screening.

[0021] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of Pulsatilla saponin B4 in the preparation of antimalarial drugs.

2. The application according to claim 1, characterized in that, The antimalarial drugs exert their antimalarial effect by modulating the function of the host's immune system.

3. The application according to claim 1, characterized in that, The antimalarial drug includes at least one of the following: improving weight loss caused by malaria infection, peripheral blood parasitemia, improving survival rate after infection, or reducing immunopathological damage caused by malaria infection.

4. The application according to claim 3, characterized in that, The immunopathological injury includes at least one of the following: a)-g) a) Splenomegaly; b) Damage to the spleen's tissue structure; c) Increased erythrocyte adhesion in liver blood vessels; d) Increased white blood cell recruitment; e) Malaria pigment deposition; f) Damage to the thymus structure; g) Disruption of lymph node structure.

5. The application according to any one of claims 1-4, characterized in that, The antimalarial drugs used to modulate the immune response of hosts infected with malaria include at least one of the following: a)-c). a) Promotes T cell and / or B cell activation; b) Regulate macrophage function; c) Promotes the activation of the interferon signaling pathway.

6. The application according to claim 5, characterized in that, The promotion of T cell and / or B cell activation is manifested by increased mRNA expression levels of Cd3 and / or Cd19 in the spleen; Macrophage function is regulated by increased mRNA expression levels of Nos1, Tnf, and II6 and / or decreased mRNA expression levels of Arg1 in the spleen. The activation of the interferon signaling pathway is promoted by increased serum IFN-γ levels and / or increased mRNA expression levels of II12 and Cxcl9 in the spleen.

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