Application of vitamin D in preparation of medicine for preventing and / or treating echinococcosis infection
High-dose vitamin D treatment addresses the immune evasion of echinococcal cysts by regulating gut microbiota, specifically reducing lactobacilli abundance, effectively inhibiting cyst growth and proliferation.
Patent Information
- Application Number
- CN202510507492.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-15
AI Technical Summary
Existing drugs for treating echinococcal infection, such as albendazole, are not effective and are difficult to effectively inhibit parasite metabolism and immune escape.
Drugs are prepared using high-dose vitamin D (not less than 1000 IU/kg or 100 IU/mL). By regulating the abundance of Lactobacillus in the intestinal flora, it affects the host immune environment and inhibits the parasite of echinococcosis.
High doses of vitamin D significantly reduce the weight of echinococcal cysts and inhibit the growth of parasites, providing a new strategy for treating echinococcal infection.
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Figure CN120305270A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of vitamin D(V D ) in the preparation of a drug for preventing and / or treating echinococcus infection. Background Art
[0002] Echinococcus infection can form chronic inflammatory lesions with cysts as the core in the host liver. In this microenvironment, immune cells (such as macrophages, Th2 cells) interact with antigens secreted by parasites to form an immunosuppressive barrier, leading to parasite immune escape and continuous proliferation of cysts. Existing treatment methods, such as albendazole, can inhibit the metabolism of parasites to a certain extent, but the effect is not good. Summary of the Invention
[0003] In view of this, the purpose of the present invention is to provide the application of vitamin D in the preparation of a drug for preventing and / or treating echinococcus infection.
[0004] The present invention provides the application of vitamin D in the preparation of a drug for preventing and / or treating echinococcus infection, and the concentration of vitamin D in the drug is not less than 1000 IU / kg or 100 IU / mL.
[0005] Preferably, the echinococcus infection includes alveolar echinococcus infection and / or multilocular echinococcus infection.
[0006] Preferably, the multilocular echinococcus infection includes at least one of the following diseases: hepatic alveolar echinococcosis, pulmonary alveolar echinococcosis, and cerebral alveolar echinococcosis.
[0007] Preferably, the echinococcus infection includes an increase in the weight and / or number of echinococcus cysts and / or an increase in the abundance of intestinal lactobacilli.
[0008] Preferably, the vitamin D includes at least one of the following forms: vitamin D tablets, vitamin D drops, and multivitamin tablets containing vitamin D.
[0009] Preferably, the vitamin D includes at least one of the following salt forms: vitamin D2 gluconate, vitamin D3 cholesteryl ester, vitamin D3 calcium carbonate, and vitamin D3 palmitate.
[0010] Preferably, the drug further includes pharmaceutically acceptable excipients;
[0011] The pharmaceutically acceptable excipients include at least one of the following: fillers, diluents, binders, wetting agents, flavoring agents, antioxidants, disintegrants, lubricants, glidants, effervescent agents, coloring agents, dispersants, film formers, plasticizers, pore-forming agents, light-blocking agents, pH adjusters, osmotic pressure regulators, chelating agents, and thickening agents.
[0012] Preferably, the dosage form of the drug includes at least one of the following: powder, tablet, capsule, pill, and oral liquid.
[0013] Preferably, the drug efficacy also includes regulating the abundance of Lactobacillus in the intestinal flora.
[0014] Preferably, the drug achieves the purpose of preventing and treating echinococcus infection by reducing the abundance of Lactobacillus in the intestinal flora.
[0015] The present invention provides the use of vitamin D in the preparation of a drug for preventing and / or treating echinococcus infection. Experiments show that Lactobacillus in the animal intestine mediates the parasitism of echinococcus, and the abundance of Lactobacillus is positively correlated with the echinococcus cyst; and the present invention uses a high dose (10000 IU / kg) of V D Oral administration can effectively reduce the weight of echinococcus cysts in animals, thereby achieving the inhibition of the parasitism of Echinococcus multilocularis. At the same time, compared with the V D Normal dose (1000 IU / kg) feeding group, the Lactobacillus in the feces of mice in the high-dose feeding group (10000 IU / kg) of V D Significantly decreased, indicating that high-dose V D May affect the host immune environment by changing the composition of the intestinal flora, thereby inhibiting the parasitism of Echinococcus multilocularis. The present invention provides new experimental evidence for the potential role of V D In regulating Echinococcus multilocularis infection, and provides new ideas and strategies for continuing to explore the mechanism of action of V D On the impact of parasite infection through immune regulation and intestinal microecological regulation. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Effects of the flora induced by Echinococcus multilocularis infection on immune cells, where a is the fecal microbiota transplantation (FMT) process of mice and control mice after 12 weeks of Echinococcus multilocularis infection; b is the differentiation result of Treg cells in the lamina propria of the colon of FMT recipient mice (CD4 + Fxop3 + Helios - ); c is the qPCR detection result of the IL-10 expression level in the colon of FMT recipient mice; d is the statistical result of the cyst size and weight of FMT recipient mice after reinfection with Echinococcus multilocularis for 8 weeks; e is the correlation result between the Lactobacillus abundance and cyst weight in the feces of mice infected with Echinococcus multilocularis.
[0017] Figure 2 For the results of Lefse analysis in 16S rRNA sequencing of intestinal contents of C57 mice after 12 weeks of infection with Echinococcus multilocularis;
[0018] Figure 3 Standard curve for the measured abundance of Lactobacillus
[0019] Figure 4 For different doses of V D Detection results of Lactobacillus abundance in feces of mice infected with Echinococcus multilocularis for 12 weeks after feeding
[0020] Figure 5 For different doses of V D Statistical results of cyst weight of mice infected with Echinococcus multilocularis for 12 weeks after feeding Specific implementation mode
[0021] The present invention provides the application of vitamin D in the preparation of drugs for preventing and / or treating Echinococcus infection, and the concentration of vitamin D in the drug is not less than 1000 IU / g or 100 IU / mL.
[0022] In the present invention, the Echinococcus infection preferably includes Echinococcus granulosus infection and / or Echinococcus multilocularis infection. Echinococcus granulosus infection is caused by Echinococcus granulosus, and this infection forms single or multiple vesicles called Echinococcus cysts, and these vesicles contain protoscolices inside. Echinococcus multilocularis infection is a lesion caused by Echinococcus multilocularis. Specifically, multiple small Echinococcus cysts are connected to each other without obvious cyst wall separation, and this lesion is also called alveolar echinococcosis. According to different infection sites, the Echinococcus multilocularis infection preferably includes at least one of the following diseases: hepatic alveolar echinococcosis, pulmonary alveolar echinococcosis, and cerebral alveolar echinococcosis. The Echinococcus infection preferably includes an increase in the weight and / or number of Echinococcus cysts and / or an increase in the abundance of Lactobacillus in the intestine. The efficacy of the drug preferably also includes regulating the abundance of Lactobacillus in the intestinal flora. The drug preferably achieves the purpose of preventing and treating Echinococcus infection by reducing the abundance of Lactobacillus in the intestinal flora.
[0023] In an embodiment of the present invention, the flora infected with Echinococcus multilocularis was inoculated into mice. After 12 weeks, the intestinal flora of the mice changed. Compared with the control group, the abundance of Lactobacillus increased, and at the same time, the cyst weight of Echinococcus multilocularis increased significantly. At the same time, through correlation analysis, it was found that there was a positive correlation between the increase in the abundance of Lactobacillus and the cyst weight of Echinococcus multilocularis. The effects of vitamin D in anti-Echinococcus infection were evaluated by high-dose and normal-dose administration of vitamin D. The results showed that compared with the normal-dose administration group, high-dose administration of vitamin D could effectively reduce the cyst weight of Echinococcus and at the same time inhibit the abundance of Lactobacillus, indicating that high-dose administration of VD may improve the immune response and reduce the survival and proliferation of Echinococcus multilocularis in mice by regulating the level of Lactobacillus in the intestinal flora.
[0024] In the present invention, the vitamin D preferably includes at least one of the following forms: vitamin D tablets, vitamin D drops, and multivitamin tablets containing vitamin D. Since the vitamin D generally includes vitamin D2 and / or vitamin D3, therefore, the vitamin D preferably includes at least one of the following salt forms: vitamin D2 gluconate, vitamin D3 cholesterol salt, vitamin D3 calcium carbonate salt, and vitamin D3 palmitate. The present invention has no special limitation on the source of the vitamin D, and the well-known vitamin D in the art can be used. In the examples of the present invention, the vitamin D is purchased from Tianjin Keao Xieli Feed Co., Ltd., and the product number is 2024042922.
[0025] In the present invention, the drug preferably further comprises pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients preferably include at least one of the following: fillers, diluents, binders, wetting agents, flavoring agents, antioxidants, disintegrants, lubricants, glidants, effervescent agents, colorants, dispersants, film formers, plasticizers, pore formers, light blockers, pH adjusters, osmotic pressure regulators, chelating agents, and thickeners. The filler is also called diluent and is used to fill the volume and weight of tablets or capsules, thereby diluting the main drug to facilitate tabletting and filling. The binder is used to increase the mechanical strength of the tablets so that they can better maintain their overall shape. The disintegrant is used to rapidly disintegrate and dissolve the drug in the gastrointestinal tract so that the drug can be better absorbed. The lubricant can be used to reduce the friction between drug particles and make the preparation flow more easily. The glidant is used to increase the fluidity of the preparation and improve the uniformity and compressibility of the preparation. The wetting agent is used to better contact the solid drug with the liquid to promote dissolution and absorption. The effervescent agent is used to generate bubbles in the body to promote the dissolution and release of the drug. The colorant is used to impart a specific color to the drug for identification and differentiation of different preparations. The sweetening agent is used to improve the taste of the drug. The preservative is used to protect the chemical structure of the drug and extend the shelf life of the drug. The dispersant is used to improve the dispersibility of the drug. The film former is used to prepare film-controlled release preparations. The plasticizer is used to increase the flexibility and plasticity of the material. The pore former is used to prepare porous matrix tablets to control drug release. The light blocker is used to prevent the influence of light on the drug. The pH adjuster is used to adjust the pH value of the preparation to adapt to the optimal release conditions of the drug or improve the compliance of patients. The osmotic pressure regulator is used to regulate the osmotic pressure of the preparation to prevent hemolysis or affect drug absorption. The chelating agent is used to stabilize the drug solution and prevent aggregation of drug molecules. The permeation enhancer is used to enhance the ability of the drug to penetrate biological membranes and improve the bioavailability of the drug. The thickener is used to increase the viscosity of the preparation and control the drug release rate. The present invention does not impose special restrictions on the types of the above excipients, and the types of excipients well-known in the art can be used. The dosage form of the drug preferably includes at least one of the following: powder, tablet, capsule, pill, and oral liquid. The present invention does not impose special restrictions on the preparation method of the drug, and the preparation methods of drugs well-known in the art can be used. In the drug, the concentration of vitamin D is preferably 1200-10000 IU / kg or 120-1000 IU / mL, and more preferably 150-8000 IU / g or 150-800 IU / mL. The recommended dose of the drug for a person is 1000-50000 IU per day, and the specific dose taken according to body weight is 50-800 IU / kg / day. The dose can be appropriately adjusted according to the patient's age, weight, disease severity, and doctor's advice.
[0026] The application of vitamin D provided by the present invention in the preparation of a drug for preventing and / or treating echinococcus infection will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0027] Example 1
[0028] In this example, healthy mice were treated with ABX (antibiotic cocktail method) to prepare PGF mice, and the flora after 12 weeks of echinococcus multilocularis infection was transplanted into recipient mice by fecal microbiota transplantation (FMT) (denoted as IN-TMT, the process is shown in Figure 1 a) in, and at the same time, PGF mice (N-FMT) were used as the control group. The mice were sacrificed 4 weeks after FMT, and the colon tissues were collected. The differentiation of Treg cells in the colonic lamina propria was detected by flow cytometry ( Figure 1 b) in, and the expression level of IL-10 was detected by qPCR method. The specific steps were as follows: TRIzol reagent (product number: 15596018, Invitrogen) was used to extract total RNA from the tissues. After extraction, the RNA concentration was measured by Qubit4. After quantifying the RNA, the GoScript reverse transcription system kit (product number: A5001, Promega) and Oligo(dT) primers were used to reverse transcribe the mRNA in the total RNA of the colon tissues into cDNA. Using IL-10 primers: IL-10-F1: 5'-TTCTTTCAAACAAAGGACCAGC-3' (SEQ ID NO:1), IL-10-R1: 5'-GCAACCCAAGTAACCCTTAAAG-3' (SEQ ID NO:2) and B2M primers: B2M-F1: 5'-TTCTGGTGCTTGTCTCACTGA-3' (SEQ ID NO:3), 5'-CAGTATGTTCGGCTTCCCATTC-3' (SEQ ID NO:4) for qPCR amplification. The reaction program used cDNA as the template, and the reaction system was shown in Table 1. The amplification program was the same as above. The relative expression level was calculated by the 2 -ΔΔCT method, and normalization analysis was performed using the internal reference gene. The results are shown in Figure 1 c.
[0029] In addition, the recipient mice of FMT prepared according to the above process were infected with echinococcus multilocularis again. After 8 weeks of infection, the mice were sacrificed, and the cysts were collected. The weight of the echinococcus multilocularis cysts infected by the recipient mice was weighed using a precision electronic balance ( Figure 1 d) in; at the same time, the abundance of Lactobacillus in the feces of the donor mice infected with echinococcus multilocularis and the cyst weight were detected, and the Spearman correlation coefficient analysis method was used to analyze the correlation between the cyst weight and the abundance of Lactobacillus in the feces ( Figure 1 e) in.
[0030] The results of intestinal flora abundance determination are shown in Figure 2 . The study found that after 12 weeks of infection with Echinococcus multilocularis, the intestinal flora of mice changed significantly, and the abundance of Lactobacillus (g_Lactobacillus and f_Lactobacillaceae) increased significantly.
[0031] In addition, the proportion of CD4 + Foxp3 + Heliso - cells in the colon of recipient mice transplanted with feces from the infected group was significantly higher than that of recipient mice transplanted with feces from the non-infected group ( Figure 1 b) in Figure 1 c)), and the expression level of IL-10 in the colon tissue also increased significantly ( Figure 1 c)). When the recipient mice after FMT were infected with Echinococcus multilocularis, it was found that the cysts of the recipient mice transplanted with feces from the infected group were larger after infection with Echinococcus multilocularis ( Figure 1 d)). And through correlation analysis, it was found that the abundance of Lactobacillus in the feces of donor mice was significantly positively correlated with the weight of the cysts ( Figure 1 e)). The above results prove that Lactobacillus in the intestinal microbiota can mediate the immune escape process of Echinococcus multilocularis.
[0032] Example 2
[0033] Application of vitamin D (V D ) in anti-Echinococcus multilocularis infection
[0034] 1. Establishment and grouping of animal models
[0035] Animals: Healthy SPF-grade C57BL / 6J female mice (Vital River, license SCXK Jing 2021-0006), weighing 20±2 g.
[0036] Grouping: According to the cross design of V D dose (0, 1000 IU / kg, 10000 IU / kg) × Echinococcus multilocularis infection, with at least 6 mice in each group.
[0037] Feed: Sterile irradiated feed (Keao Xieli, batch number KAXL202301VD), and the V D content was verified by HPLC (error <5%).
[0038] 2. Specific steps
[0039] (1) After feeding with different doses of V D for 4 weeks, the mice were infected with Echinococcus multilocularis.
[0040] (2) Inject a suspension of 2000 Echinococcus multilocularis protoscoleces via intraperitoneal injection. The control group is injected with the same dose
[0041] PBS.
[0042] (3) Infect for 12 weeks.
[0043] 3. Index detection
[0044] (1) Microbiota detection:
[0045] Extract fecal genomic DNA using a DNA extraction kit (QIAGEN, QIAamp DNA Stool Kit). After extraction, measure the DNA concentration with Qubit4. After quantifying the DNA, perform qPCR amplification using specific primers for Lactobacillus (LabF362: 5'-AGCAGTAGGGAATCTTCCA-3', SEQ ID NO:5; LabR677: 5'-CACCGCTACACATGGAG-3', SEQ ID NO:6). The reaction program uses DNA as a template, performs PCR amplification with specific primers, and uses the two-step method for qPCR detection. The reaction system is as shown in Table 1 below.
[0046] Table 1 Reaction system
[0047] GoTaqqPCRMix(2x) 10 μL Forward primer (10 μM) 0.4 μL Reverse primer (10 μM) 0.4 μL DNA template 2 μL (100 ng) Nuclease-Free water 7.2 μL Total volume 20 μL
[0048] The amplification program includes: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 60°C for 1 min, for 40 cycles. The melting curve is set at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. In the final experimental data analysis, a standard curve ( Figure 3 ) is plotted based on the DNA concentration after different dilutions of the bacterial solution and its corresponding Ct value (threshold cycle number), and calculations are performed based on this to compare the changes in the relative abundances of Enterobacter and Lactobacillus in the experimental group and the control group.
[0049] (2) Cyst measurement
[0050] Decapitate the mice and collect the Echinococcus multilocularis cysts for weighing (g).
[0051] 4. Data analysis:
[0052] (1) Comparison between groups: Use Prism 9 software (GraphPad Software, La Jolla, CA) for statistical analysis. Compare the abundance differences among multiple groups through one-way analysis of variance (ANOVA). The significance level is set at p < 0.05. The data are presented as mean ± standard deviation (Mean ± SD).
[0053] (2) Correlation analysis: The Pearson correlation coefficient (r) was used to quantify the association strength between the abundance of Lactobacillus and the cyst weight.
[0054] 5. Experimental results:
[0055] (1) Microbiota regulation effect: Compared with the normal-dose (1000 IU / kg) diet-fed group, the Lactobacillus in the feces of mice in the high-dose diet-fed group (10000 IU / kg) decreased significantly, while there was no obvious change in the low-dose group ( D ( D ). In the high-dose diet-fed group (10000 IU / kg), the abundance of Lactobacillus in mice decreased significantly, showing a consistent trend with the decrease in cyst weight. It is suggested that D the high-dose diet may affect the host immune environment by changing the composition of the intestinal microbiota, thereby inhibiting the parasitism of Echinococcus multilocularis. While in the normal-dose diet-fed group (1000 IU / kg), no similar changes occurred, indicating that Figure 4 high-dose feeding may play a more obvious role in regulating the intestinal microbiota. D (2) Anti-parasitic effect: It is worth noting that D compared with the normal diet dose (1000 IU / kg) diet-fed group, the cyst weight of mice in the high-dose diet-fed group (10000 IU / kg) decreased significantly ( D ). Analysis found that D the cyst weight of the high-dose feeding group decreased significantly, indicating that high-dose
[0056] diet feeding may inhibit the growth of parasites through immunomodulatory effects. Combining the results of Example 1 that the abundance of Lactobacillus was positively correlated with the cyst weight, it further verified the important role of changes in the intestinal microbiota in Echinococcus multilocularis infection. High-dose D feeding may improve the immune response and reduce the survival and proliferation of Echinococcus multilocularis in mice by regulating the level of Lactobacillus in the intestinal microbiota. These results provide new experimental evidence for the potential role of D in regulating Echinococcus multilocularis infection, and continue to explore the Figure 5 influence mechanism of D on parasite infection through immunomodulation and intestinal microecological regulation, providing new ideas and strategies for the treatment of Echinococcus multilocularis. D D D D
[0057] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. Use of vitamin D in the preparation of a drug for preventing and / or treating echinococcus infection, wherein the concentration of vitamin D in the drug is not less than 1000 IU / kg or 100 IU / mL.
2. The application according to claim 1, characterized in that, The echinococcus infection includes alveolar echinococcus infection and / or multilocular echinococcus infection.
3. The application according to claim 2, wherein The multilocular echinococcus infection includes at least one of the following diseases: hepatic alveolar echinococcosis, pulmonary alveolar echinococcosis, and cerebral alveolar echinococcosis.
4. The application according to claim 1, characterized in that The echinococcus infection includes an increase in the weight and / or number of echinococcus cysts and / or an increase in the abundance of intestinal lactobacilli.
5. The application according to claim 1, characterized in that The vitamin D includes at least one of the following forms: vitamin D tablets, vitamin D drops, and multivitamin tablets containing vitamin D.
6. The application according to claim 1, wherein The vitamin D includes at least one of the following salt forms: vitamin D2 gluconate, vitamin D3 cholesteryl ester, vitamin D3 calcium carbonate, and vitamin D3 palmitate.
7. The application according to any one of claims 1 to 6, characterized in that The drug further includes a pharmaceutically acceptable excipient; The pharmaceutically acceptable excipient includes at least one of the following: filler, diluent, binder, wetting agent, flavoring agent, antioxidant, disintegrant, lubricant, glidant, effervescent agent, colorant, dispersant, film-forming agent, plasticizer, pore-forming agent, opacifying agent, pH adjuster, osmotic pressure regulator, chelating agent, and thickening agent.
8. The application according to claim 7, wherein The dosage form of the drug includes at least one of the following: powder, tablet, capsule, pill, and oral liquid.
9. The application according to claim 1, characterized in that, The efficacy of the drug also includes regulating the abundance of lactobacilli in the intestinal flora.
10. The application according to claim 9, wherein The drug achieves the purpose of preventing and treating echinococcus infection by reducing the abundance of lactobacilli in the intestinal flora.