Application of rutin in preparation of preparation for preventing and / or treating talaromyces marneffei infection
By using rutin to regulate the host immune response, the problems of nephrotoxicity, drug resistance risk and low accessibility of existing anti-Marnifera Basilica infection drugs have been solved, and safer and more effective therapeutic effects have been achieved.
Patent Information
- Application Number
- CN202510648875.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-20
- Publication Date
- 2025-06-27
AI Technical Summary
The existing anti-Marnifer basket bacteria infection drugs have problems such as nephrotoxicity, drug resistance risk and low accessibility, resulting in limited treatment safety and effectiveness.
Rutin is used as a drug for anti-T.marneffei infection, and inhibit infection and reduce fungal load by regulating host immune response, especially key transcription factors such as IL-17 signaling pathway and AP-1.
Rutin significantly reduces fungal load, reduces side effects and risk of drug-resistant strain generation, and improves treatment safety and survival in mice.
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Figure CN120204250A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of rutin in the preparation of a preparation for preventing and / or treating Talaromyces marneffei infection. Background Art
[0002] Talaromyces marneffei (abbreviated as T. marneffei or Tm) is a dimorphic fungus that mainly infects immunocompromised individuals and can cause severe systemic fungal infections. The infection rate of this disease is relatively high in HIV / AIDS patients and has become the third most common opportunistic infection after tuberculosis and cryptococcal infection. Studies in Guangxi, China, have shown that the mortality rate of HIV patients co-infected with T. marneffei is significantly higher than that of non-infected patients. Moreover, with the increase in cancer, autoimmune diseases, and organ transplant patients, the number of T. marneffei infection cases in non-HIV-infected individuals is on the rise, and the mortality rate is relatively high. Since this disease has not been included in the routine monitoring system, the actual infection burden may be severely underestimated, and the World Health Organization (WHO) has listed it as a medium-priority pathogen in the "List of Priority Fungal Pathogens".
[0003] Currently, the treatment regimens for T. marneffei infection mainly rely on amphotericin B and azole antifungal drugs. However, amphotericin B has serious adverse reactions such as nephrotoxicity, electrolyte disorders, and bone marrow suppression, which limit its clinical application. Moreover, its liposomal modified preparation has low accessibility in some Asian countries due to high cost and limited supply. Although azole drugs have relatively low toxicity and side effects, they require long-term liver function monitoring and carry the risk of drug resistance. Given the limitations of existing drugs, the development of new, highly effective, low-toxic, and more accessible antifungal drugs has become an urgent need. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide the application of rutin in the preparation of a preparation for preventing and / or treating Talaromyces marneffei infection. When rutin is applied to anti-T. marneffei infection, it can effectively reduce side effects, lower the risk of generating drug-resistant strains, and improve the safety of treatment.
[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:
[0006] The present invention provides the application of rutin in the preparation of a preparation for preventing and / or treating Talaromyces marneffei infection.
[0007] Preferably, the concentration of the rutin is 20 - 160 μM.
[0008] Preferably, the usage mode of the rutin is injection.
[0009] The present invention also provides a preparation for preventing and / or treating Talaromyces marneffei infection, comprising rutin and a pharmaceutically acceptable carrier.
[0010] The present invention also provides the use of rutin in the preparation of a preparation for regulating the host immune response.
[0011] The present invention also provides a preparation for regulating the host immune response, comprising rutin and a pharmaceutically acceptable carrier.
[0012] The present invention also provides the use of rutin in the preparation of a preparation for reducing fungal burden.
[0013] The present invention also provides a preparation for reducing fungal burden, comprising rutin and a pharmaceutically acceptable carrier.
[0014] Beneficial effects:
[0015] (1) The present invention for the first time discovers that the natural flavonoid compound rutin has a significant inhibitory effect on T. marneffei infection. In the THP-1 macrophage infection model at the cellular level, the compound-T. marneffei co-culture experiment, and the mouse infection model of the animal model, it shows that rutin can significantly reduce the fungal burden and alleviate inflammatory damage; compared with the existing anti-T. marneffei drugs mainly relying on amphotericin B, itraconazole, etc. that act by destroying the fungal cell membrane, rutin inhibits infection by regulating the host immune response, providing a brand-new anti-T. marneffei strategy, and it is dose-dependent. Although the antibacterial effect is not as good as that of amphotericin B, it has lower toxicity and can effectively improve the survival rate of mice;
[0016] (2) The present invention reveals that rutin regulates the process of T. marneffei infection through the IL-17 signaling pathway and key transcription factors such as AP-1, and produces corresponding dynamic regulation at different stages of infection; RNA-Seq analysis shows that rutin can enhance the host immune response by upregulating Jund and chemokines cxcl1 / cxcl2, and inhibit inflammation-related genes such as Jund in the late stage of infection to reduce excessive inflammatory damage; this mechanism is different from the direct antibacterial effect of traditional antifungal drugs, providing a new target and action strategy for anti-T. marneffei infection, and opening up a new idea for antifungal treatment targeting host-pathogen interaction;
[0017] (3) As a natural flavonoid compound, rutin has good biological safety; compared with the liver and kidney toxicity and drug resistance problems that may be caused by conventional antifungal drugs such as amphotericin B and itraconazole, the application of rutin in anti-T. marneffei infection can effectively reduce side effects, reduce the risk of producing drug-resistant strains, and improve the treatment safety. Description of the drawings
[0018] Figure 1 Results of direct anti-T. marneffei activity detection of rutin;
[0019] Figure 2 Results of body weight and liver and spleen organ indices of mice in animal experiments;
[0020] Figure 3 Results of anti-T. marneffei activity detection of rutin in cell experiments;
[0021] Figure 4 Results of anti-T. marneffei activity detection of rutin in animal experiments;
[0022] Figure 5 Transcription levels of transcription factors Jund, cxcl1, and cxcl2 in the IL-17 pathway in the liver and spleen of mice 7 days after T. marneffei infection. Among them, A is the transcription level of Jund in the mouse liver, B is the transcription level of cxcl1 in the mouse liver, C is the transcription level of cxcl2 in the mouse liver, D is the transcription level of Jund in the mouse spleen, E is the transcription level of cxcl1 in the mouse spleen, and F is the transcription level of cxcl2 in the mouse spleen;
[0023] Figure 6 Transcription levels of transcription factors Jund, cxcl1, and cxcl2 in the IL-17 pathway in the liver and spleen of mice 14 days after T. marneffei infection. Among them, A is the transcription level of Jund in the mouse liver, B is the transcription level of cxcl1 in the mouse liver, C is the transcription level of cxcl2 in the mouse liver, D is the transcription level of Jund in the mouse spleen, E is the transcription level of cxcl1 in the mouse spleen, and F is the transcription level of cxcl2 in the mouse spleen. Detailed implementation methods
[0024] The present invention provides the use of rutin in the preparation of a preparation for preventing and / or treating Talaromyces marneffei infection.
[0025] In the present invention, the concentration of the rutin is 20 - 160 μM, preferably 60 - 120 μM, and further preferably 90 μM.
[0026] In the present invention, the rutin is used by injection.
[0027] The present invention also provides a preparation for preventing and / or treating Talaromyces marneffei infection, comprising rutin and a pharmaceutically acceptable carrier.
[0028] In the present invention, the preparation is preferably an injection containing rutin.
[0029] The present invention also provides the use of rutin in preparing a preparation for regulating host immune response.
[0030] The present invention also provides a preparation for regulating host immune response, comprising rutin and a pharmaceutically acceptable carrier.
[0031] The present invention also provides the use of rutin in preparing a preparation for reducing fungal load.
[0032] The present invention also provides a preparation for reducing fungal load, comprising rutin and a pharmaceutically acceptable carrier.
[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0034] Example 1
[0035] Preparation of PDA medium: After washing the conical flask twice with ultrapure water (UP water), add 20.05g potato dextrose agar (PDA) medium into 500mL UP water, mix thoroughly, wrap the bottle mouth with kraft paper to prevent contamination, and sterilize at 121℃ for 20min. At the same time, place the culture dish and other consumables in the biosafety cabinet for ultraviolet sterilization for 30min. After the culture medium is cooled to 60℃, the surface is disinfected and 5μl of penicillin-streptomycin mixture (purchased from Beijing Solebow Technology Co., Ltd.) is added to prevent bacterial contamination. After mixing, pour into the culture dish to 3 / 4 volume, shake gently to spread evenly, open the lid of the culture dish and irradiate with ultraviolet light for 30min to reduce contamination. The culture medium is prepared and used immediately.
[0036] Preparation of YPD medium: Wash the conical flask twice with UP water, prepare YPD medium at 40.10g / L, cover after mixing, wrap the bottle mouth with kraft paper, and sterilize at 121℃ for 20min. At the same time, place the culture dish and other consumables in the biosafety cabinet for UV sterilization for 30min; after the culture medium is cooled to 50℃, disinfect it with 75% alcohol and move it into the biosafety cabinet, add penicillin-streptomycin mixture, mix well and pour into the culture dish about three-quarters of the volume, shake gently; sterilize with UV for 30min to ensure solidification and reduce contamination;
[0037] T. marneffei spore expansion culture: Take 10 μl of conidia suspension (purchased from Beina Biotechnology) and add it dropwise to PDA medium. After sealing, place it in a 27°C incubator and culture it for 2 weeks. The back of the medium will be rose red and the front will be grayish white powder, which is T. marneffei conidia.
[0038] T. marneffei conidia collection: Scrape the T. marneffei conidia on the culture surface with an inoculation loop and place them in a sterile PBS centrifuge tube to obtain a spore suspension; slowly drip the spore suspension into a 50 mL centrifuge tube lined with sterile glass wool to filter the mycelium and impurities and obtain a conidia suspension; take 3 drops of the conidia suspension and drop them into a 6-well plate, add 1 mL of DMEM culture medium, culture for 24 h and 48 h to observe for contamination. After confirming no contamination, aliquot in a timely manner and store at 4 °C;
[0039] Construction of a THP-1 macrophage model infected with T. marneffei: Inoculate THP-1 cells (purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) into a 48-well cell culture plate (200,000 cells per well), and use phorbol 12-myristate 13-acetate (PMA, final concentration 50 ng / mL) to stimulate for 48 h to differentiate them into THP-1 macrophages; discard the supernatant culture medium, and respectively use complete DMEM culture medium [containing 10% inactivated fetal bovine serum, 100 U / mL penicillin-streptomycin solution] to prepare a suspension containing 0.28 μM itraconazole, 0.26 μM amphotericin B (AMB), and 80 μM rutin (dissolve rutin powder in DMSO and then dilute it to 80 μM with the culture medium), gently add it to the cell plate, and after reacting for 3 h, add T. marneffei conidia (MOI = 3, 300,000) to infect THP-1 macrophages to obtain a THP-1 macrophage model infected with T. marneffei; place it in a 37 °C, 5% CO2 cell culture incubator for culture;
[0040] Construction of a T. marneffei-infected animal model: SPF healthy female Balb / c mice [purchased from Beijing Specific Pathogen Free Biotechnology Co., Ltd., 18 - 20 g] are adaptively fed for 4 d at 18 - 20 °C and 50 - 60% humidity, and then ear-tagged;
[0041] Randomly divide the mice into 6 groups (6 mice in each group): control group (Control), T. marneffei group, Rutin 40 mg / kg group, Rutin 80 mg / kg group, AMB 0.5 mg / kg group; mice in the Rutin group are intraperitoneally injected with Rutin at doses of 40 mg / kg and 80 mg / kg continuously for 5 d; except for the control group which is injected with 200 μl of normal saline via the tail vein, the other groups are all injected with 2×10 7An infection model was constructed using T. marneffei conidia. From the day of infection, the control group and the T. marneffei group were intraperitoneally injected with 288 μl of normal saline daily. The Rutin 40 mg / kg group, the Rutin 80 mg / kg group, and the AMB 0.5 mg / kg group were injected with the corresponding concentrations of the drug, respectively, and the injection continued until the 7th and 14th days after infection, resulting in a T. marneffei-infected animal model.
[0042] Example 2 Detection of the direct anti-T. marneffei activity of rutin
[0043] In a 12-well cell culture plate, 500 μl of complete DMEM culture medium was first added to each well, and then 1 million T. marneffei conidia were inoculated. A negative control group (infected only with T. marneffei, without adding other drugs), a Rutin group, and a positive control AMB group were set up, with 4 replicates in each group. The final volume of the 12-well plate was 1 mL. Therefore, after adding 500 μl of the culture medium, the remaining 500 μl was used to prepare the drug solution. The Rutin group was set with concentration gradients of 20, 40, 60, 80, and 160 μM, and the concentration of the AMB group was 0.26 μM. The volume of the mother liquor taken for each group was calculated according to the required concentration to ensure that the final concentration after adding the drug met the design. The volume added to the DMSO group was the same as that of the 160 μM concentration in the Rutin group to maintain the consistency of the solvent control. All drugs were co-cultured with T. marneffei conidia for 72 h;
[0044] The culture medium was collected into a 1.5 mL EP tube, centrifuged at 14000 g for 5 min, the supernatant was discarded, and after resuspension with 200 μl of PBS, CFU detection was performed. The results are as Figure 1 shown;
[0045] The results showed that rutin had a direct killing effect on T. marneffei, and the effect was more significant with the increase in concentration.
[0046] Example 3 Detection of the anti-T. marneffei activity of rutin
[0047] Treatment of cell / tissue samples:
[0048] After culturing the T. marneffei-infected THP-1 macrophage model described in Example 1 for 12 h and 24 h respectively, it was washed 3 times with PBS. 250 μl of autoclaved water was added to each well to lyse the cells, which were pipetted and collected into a 1.5 mL EP tube. Another 250 μl of autoclaved water was added, and pipetting was repeated and combined. After centrifugation at 14000 g for 5 min to precipitate the fungi, the supernatant was discarded and resuspended with 200 μl of PBS, labeled as the "10 0 " stock solution for the cell experiment;
[0049] The body weights and behavioral states of the animal models infected with T. marneffei were monitored, and the results were as Figure 2 shown; on the 7th and 14th days after infection, the mice in each group were anesthetized with sodium pentobarbital, the liver and spleen were taken, washed with sterile PBS to remove blood, weighed, and the organ indices were calculated (organ index = organ weight / body weight), and the results were as Figure 2 shown; 30 mg of tissue was cut, resuspended in 200 μl of pre-cooled PBS, ground evenly at 4°C, and labeled as the "10 0 " stock solution for animal experiments;
[0050] During the sampling process, it was placed on ice throughout, and the samples were immediately stored at -80°C;
[0051] Fungal burden detection: Three EP tubes were labeled as "10 -1 ", "10 -2 ", "10 -3 " respectively, and each was added with 180 μl of PBS; the "10 0 " stock solution was serially diluted: 20 μl of the "10 0 " stock solution was added to the "10 -1 " tube and mixed evenly to obtain a 10-fold dilution; 20 μl of the 10-fold dilution was added to the "10 -2 " tube and mixed evenly to obtain a 100-fold dilution; 20 μl of the 100-fold dilution was added to the "10 -3 " tube and mixed evenly to obtain a 1000-fold dilution; 5 μl of each gradient dilution was added dropwise to the YPD medium, and 3 replicate points were set for each concentration; the edge was sealed with a sealing film, cultured at 37°C for 48 h, photographed to count the colonies, and the number of colonies was calculated, and the results were as Figures 3 - 4 shown;
[0052] Among them, the number of colonies in cell experiments = colony count × dilution factor × 40,
[0053] the number of colonies in animal experiments = colony count × dilution factor × 40 × (mouse organ weight mg / 30 mg);
[0054] The results showed that Rutin had obvious anti-T. marneffei activity, and the longer the action time, the more significant the effect; Rutin could effectively reduce the fungal burden in the liver and spleen tissues, and its effect increased with the increase of the dose.
[0055] Example 4 Real-time fluorescence quantitative PCR detection of the expression of transcription factors and chemokines
[0056] RNA extraction from mouse tissues: Cut 25 mg of mouse liver and spleen tissues. After washing away blood with PBS, add 500 μl of Trizol and grind. Let it stand at room temperature for 5 min for lysis; then add chloroform (Trizol:chloroform = 5:1), vortex and mix well, incubate at room temperature for 3 min, centrifuge at 4 °C and 12,000 g for 15 min; aspirate the upper aqueous phase into a new EP tube, add an equal volume of isopropanol to precipitate RNA, let it stand at room temperature for 10 min, centrifuge at 4 °C and 12,000 g for 10 min; discard the supernatant, wash twice with 75% ethanol, centrifuge at 4 °C and 12,000 g for 5 min each time. After removing the residual ethanol, add 50 μl of DEPC water to dissolve RNA, incubate at 56 °C for 13 min; use NanoDrop 2000C to measure the RNA purity (A260 / A280 = 1.8 - 2.0), and based on the lowest concentration, unify the RNA concentration with RNase-free water;
[0057] Reverse transcription: Use the PrimerScriptTM RT Master Mix-RR036A kit to prepare the reaction system on ice and perform reverse transcription on RNA;
[0058] The reaction system and reaction program are shown in Tables 1 - 2;
[0059] Table 1 Reverse transcription reaction system
[0060] Reaction system configuration Required volume Primer Script Raster Mix(5×) 6.00 μl DEPC water 20.00 μl RNA(≤1000 ng) 4.00 μl Total 30.00 μl
[0061] Table 2 Reverse transcription reaction program
[0062] Steps Conditions Reaction time Step 1 37℃ 30 min Step 2 85℃ 5s Step 3 4℃ Reaction stopped, +
[0063] Primer synthesis: Design primers through Primer Bank and NCBI (as shown in Table 3), and Gapdh is used as the internal reference gene;
[0064] Table 3 Primer sequences
[0065]
[0066] qPCR detection: Use 2×RealStar Fast SYBR qPCR Mix (A304), mix well and add to a 96-well plate, seal the film, centrifuge at 1000 rpm for 2 min, and then place it in the ABI qPCR system for qPCR detection. The detection results are as Figures 5 - 6 shown;
[0067] The reaction system and reaction program for qPCR detection are shown in Tables 4 - 5;
[0068] Table 4 Reaction system for qPCR
[0069] Reaction system Required volume 2×RealStar Fast SYBR qPCR Mix 10 μl Forward primer 0.4 μl Reverse primer 0.4 μl DEPC water 8.2 μl cDNA template 1 μl Total 20 μl
[0070] Table 5 Reaction program of qPCR
[0071] Reaction conditions Reaction time 95℃ 2 min 95℃ 15s 60℃ 1 min Plate read 40 cycles Melting curve 60°C - 95°C, step 0.5°C
[0072] The results showed that in the acute infection period (7 days), rutin significantly up-regulated the expression of the key transcription factor Jund of the IL-17 pathway and the downstream chemokines cxcl1 and cxcl2 in a dose-dependent manner; while in the chronic period (14 days), rutin promoted the negative feedback regulation of host inflammation and significantly down-regulated the expression of the key transcription factor Jund of the IL-17 pathway and the downstream chemokines cxcl1 and cxcl2, also in a dose-dependent manner. It can be seen that rutin can enhance the host immune response by up-regulating Jund and chemokines cxcl1 / cxcl2, and inhibit inflammation-related genes such as Jund in the late stage of infection to reduce excessive inflammatory damage.
[0073] As can be seen from the above examples, the present invention provides the application of rutin in the preparation of a preparation for preventing and / or treating Talaromyces marneffei infection. The present invention for the first time discovers that the natural flavonoid rutin has a significant inhibitory effect on T. marneffei infection. Compared with the possible liver and kidney toxicity and drug resistance problems caused by conventional antifungal drugs such as amphotericin B and itraconazole, the application of rutin in anti-T. marneffei infection can effectively reduce side effects, reduce the risk of generating drug-resistant strains, and improve the safety of treatment.
[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. Use of rutin in the preparation of a preparation for preventing and / or treating Talaromyces marneffei infection.
2. The use according to claim 1, characterized in that: The concentration of the rutin is 20-160 μM.
3. The use according to claim 2, characterized in that: The rutin is used by injection.
4. A preparation for preventing and / or treating Talaromyces marneffei infection, characterized in that: The invention comprises rutin and a pharmaceutically acceptable carrier.
5. The application of rutin in the preparation of preparations for regulating host immune response.
6. A preparation for regulating host immune response, characterized in that The invention comprises rutin and a pharmaceutically acceptable carrier.
7. Use of rutin in the preparation of preparations for reducing fungal load.
8. A preparation for reducing fungal load, characterized in that The invention comprises rutin and a pharmaceutically acceptable carrier.