CN104069128A - Application of poly vinyl pyrrolidone (PVP) modified nano-silver and imidazole medicines in preparation of drug tolerance resistant fungus active combined medicines - Google Patents
Application of poly vinyl pyrrolidone (PVP) modified nano-silver and imidazole medicines in preparation of drug tolerance resistant fungus active combined medicines Download PDFInfo
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Abstract
The invention provides an application of poly vinyl pyrrolidone (PVP) modified nano-silver and imidazole medicines in preparation of drug tolerance resistant fungus active combined medicines. The invention shows that fluconazole or voriconazole and PVP modified nano-silver are used jointly to have synergetic drug tolerance resisting fungus effect. If drug combination is carried out on the imidazole drug resistant strain CA10 (MIC>256mg/L), the minimum inhibitory concentration can be lowered to 1mg/L, and the drug resistance is reversed.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of PVP modified nano silver (Ag-NPs) and azole drugs fluconazole or voriconazole in preparation of drugs for resisting drug-resistant fungus activity combination.
Background
In recent 20 years, with the increase of patients suffering from cancer radiotherapy, chemotherapy, organ transplantation and AIDS, and the long-term wide application of broad-spectrum antibiotics and immunosuppressants, patients with low immune function are increasing, the incidence rate of deep fungal infection is rapidly increased, and the fungal infection becomes one of the main death reasons of patients with serious diseases of the low immune function. While antifungal drugs have been used in large quantities for a long time in the clinic, drug-Resistant bacteria have become the leading cause of failure of clinical antifungal therapy [ Ruhnke M, Eigler A, Tennagen I, Geiseler B, Engelmann F, Trautmann M.1994. expression of Fluonazole-Resistant Strains of Candida albicans in Patients with Recurred Oropyrophageal Candida and Human Immunodeficiency Virus infection. J Clin Microbiol.32: 2092-. Various candida diseases, cryptococcus neoformans and aspergillus fumigatus still are the most common deep pathogenic fungi. The detection rate of candida in hospital blood specimens was 7.6%, ranking the fourth place of all pathogens, the first place of pathogenic fungi. The most common pathogenic species in candida is candida albicans.
Currently, the number of antifungal drugs available for clinical selection is limited, and the commonly used antifungal drugs mainly include polyene antibiotics (amphotericin B), azole antifungal drugs (fluconazole), allylamine antifungal drugs (terbinafine), flucytosine, echinocandin antifungal compounds and the like. Among them, fluconazole is the most widely used antifungal drug in clinical application due to its good bioavailability, less adverse reactions and moderate price. However, since fluconazole only has the function of inhibiting fungi, the fungi are subjected to fast-developing drug resistance in long-term and repeated treatment. Moreover, fluconazole-resistant fungi have cross-resistance to other antifungal drugs, so that ketoconazole, itraconazole and even amphotericin B are overwhelmed by more and more resistant bacteria, which is a main reason for failure of clinical fungal infection treatment.
Strategies to combat fungal resistance are mainly: (1) developing new drugs. However, the development process of the new drug is expensive, the time is long, and the elimination rate of the candidate drug is high; (2) the medicine combination is used. The combination of antifungal drugs is one of the most convenient strategies to overcome the drug resistance of fungi clinically at present. The theoretical advantages of combination therapy are: different drugs inhibit different stages of fungal metabolism to produce synergistic effects, one drug acts on the cell wall or cell membrane to increase the penetration of the other drug into the cell; act on different molecular targets; pharmacokinetic/pharmacodynamic complementation; the single dosage is reduced, so that the toxic and side effects are reduced; reduce the occurrence of drug resistance, etc. Clinical combinations have focused on the treatment of cryptococcosis, invasive candidiasis and invasive aspergillosis.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of PVP modified nano silver and azole drugs in the preparation of drugs for resisting drug-resistant fungus activity combination.
Wherein, the azole drug is selected from one or two of fluconazole and voriconazole.
Wherein the drug-resistant fungi is drug-resistant Candida albicans.
The invention uses a standard microdilution method to determine the drug sensitivity, uses a checkerboard microdilution method, an agar diffusion method and a time-sterilization curve method to evaluate the in vitro relationship of the drug-resistant candida albicans in the combined application of Ag-NPs and fluconazole or voriconazole, and the result shows that: the Ag-NPs and fluconazole or voriconazole jointly applied to the azole-resistant strain have a synergistic action relationship.
The cytological mechanism of synergy consists in: the Ag-NPs increase the defect of the fungal cell membrane, the fluconazole and the voriconazole can increase the quantity of the Ag-NPs adhered to the fungal cell membrane, and the combination of the Ag-NPs and the azole can obviously inhibit the normal budding process and the like.
The molecular mechanism of synergy lies in: the PVP modified nano silver and azole drugs generate synergistic effect by acting on ergosterol biosynthesis pathway related genes and efflux pump expression genes.
The invention also provides a pharmaceutical composition, which comprises PVP modified nano silver and azole drugs, wherein the azole drugs are selected from one or two of fluconazole and voriconazole.
Has the advantages that: in order to overcome the drug resistance phenomenon of candida albicans, the invention provides the application of azole drugs and PVP modified nano-silver in the preparation of drugs for resisting drug-resistant fungus activity combination by utilizing the existing drug combination.
Specifically, compared with the prior art, the invention has the following outstanding advantages and effects:
(1) the invention shows that when the fluconazole or the voriconazole and the PVP modified nano silver are combined for application, the synergistic drug-resistant fungus resisting effect can be generated. For azole drug-resistant strain CA10(MIC >256mg/L), the minimum inhibitory concentration can be reduced to 1mg/L by combined administration, thereby reversing drug resistance.
(2) The invention shows that the medicine acting on ergosterol biosynthesis pathway and inhibiting cell membrane efflux pump gene expression is probably a molecular mechanism combining antifungal effect, and provides a possible research direction for new medicine development and new application of old medicines.
(3) The nano silver gel is used for preventing and treating gynecological diseases because the nano silver gel is a non-antibiotic broad-spectrum high-efficiency antibacterial agent. The nano silver modified by polyvinylpyrrolidone (PVP) has higher stability. When the compound is used together with azoles, the compound is effective on drug-resistant fungi, and the respective dosage can be reduced, so that adverse drug reactions are reduced, the application range of the drug is expanded, and the occurrence of drug resistance is slowed down.
Drawings
FIG. 1 is a characterization map of AgNPs. Wherein A is the electron microscope data of AgNPs alone or after azole drugs are added; b is AgNPs ultraviolet absorption spectrum; c is crystal diffraction data of AgNPs; d is the hydraulic diameter and zeta potential value of AgNPs alone or after azole drugs are added.
FIG. 2 is a synergistic effect analysis of AgNPs and azoles combined with drug-resistant Candida albicans CA 10. Wherein A is an equivalence curve after the fluconazole and the AgNPs are combined; b is a flat plate dropping method which suggests that the fluconazole and the AgNPs have a sterilization synergistic effect after being used together; c is an agar diffusion method experimental result; d is the sterilization curve experimental result. The strain is a drug-resistant strain CA10, and FLC represents fluconazole; VOR represents voriconazole.
FIG. 3 is a graph showing the change in the morphology of Candida albicans when fluconazole is used in combination with AgNPs. Wherein A is the adhesion effect of AgNPs on fungal cells, the concentration of fluconazole is 4mg/L, the concentration of AgNPs is 2mg/L and the combined application thereof; b is the adhesive effect of the fluconazole concentration influencing AgNPs on fungal cells; c is the comparison of relative budding rate, the concentration of fluconazole is 4mg/L, the concentration of AgNPs is 2mg/L and the combined application thereof. Sprouts were defined as cells smaller than the size of the parent 1/2.**p<0.01。
FIG. 4 is a graph showing the effect of fluconazole in combination with AgNPs on the uptake of propidium iodide by Candida albicans. Wherein A is combined drug for treating whitish ideaThe effect of Propidium Iodide (PI) uptake by the pearlobacteria; b is the ratio change of the candida albicans absorbing propidium iodide after the drug treatment. The concentration of fluconazole is 4mg/L, and the concentration of AgNPs is 4 mg/L.**p<0.01。
FIG. 5 is a graph showing the effect of quantitative PCR on gene expression of fluconazole in combination with AgNPs. The concentration of fluconazole is 4mg/L, and the concentration of AgNPs is 4 mg/L. P < 0.01.
FIG. 6 is a graph showing the effect of fluconazole in combination with AgNPs on ergosterol content. Wherein A is ergosterol ultraviolet absorption spectrum; and B is the influence of the combination of fluconazole and AgNPs on the content of ergosterol. The concentration of fluconazole is 4mg/L, and the concentration of AgNPs is 4 mg/L.**p<0.01.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The AgNPs synthesis process used in the present invention was performed with reference to literature [ Guo D, Zhu L, Huang Z, et al, anti-leukaemia activity of PVP-coated silver nanoparticles virus generation of reactive oxidative species and release of silver ions. biomaterials 2013; 34:7884-94]. The purity of the prepared AgNPs is 99.99%.
Structural characterization of AgNPs, see fig. 1:
(1) the maximum absorption peak obtained by ultraviolet full-wavelength scanning is 416 nm;
(2) the crystal diffraction data has four typical peaks of nano-silver, 111, 200, 220, 311;
(3) the particle size is counted by electron microscope observation, the AgNPs particle size distribution and the particle size after azole drugs are added have no statistical difference in polymerization degree;
(4) determining the particle size of AgNPs (1mg/mL) by a Darwen particle sizer to be 34.92 +/-2.1 nm, and adding fluconazole and voriconazole to obtain the AgNPs with the particle size of 34.67 +/-1.7 nm and 35.12 +/-2.4 nm;
(5) the zeta potential of AgNPs is-5.35 +/-1.4 mV, after 8mg/L of fluconazole and 8mg/L of voriconazole are added, the zeta potential is-4.23 +/-1.5 mV and-4.23 +/-1.2 mV, and the addition of azole drugs has no obvious influence on the stability of the AgNPs.
Example 1 in vitro synergy experiments
For in vitro susceptibility testing and checkerboard microdilution of antifungal drugs, reference is made to the National Committee for clinical Laboratory standards, methods for differentiating antibiotic disk differentiation of fungi; advanced Guideline M44-A. NCCLS, Wayne, PA, USA, 2004. The specific operation is as follows:
(1) bacterial liquid preparation and sensitivity analysis
After thawing the Candida albicans strain stored at-70 ℃, inoculating the strain to YPD solid medium, and culturing for 24h at 37 ℃. Well-developed single colonies were re-inoculated onto YPD and cultured at 35 ℃ for 24 hours to obtain pure cultures. 5 colonies with a diameter greater than 1mm were selected and made into bacterial suspension with 0.85% sterile physiological saline. Oscillating the bacterial suspension on an oscillator for 15 seconds, counting the number of bacteria by using a blood cell counting plate, and adjusting the concentration of the bacterial suspension to be 1-5 multiplied by 106cfu/ml. Diluting the strain liquid by 1000 times with RPMI-1640 culture solution to obtain inoculated strain suspension, wherein the final concentration of the strain liquid in each hole after inoculation is 0.5-2.5 multiplied by 103cfu/ml. After placing the 96-well plate in a 37 ℃ incubator for 48 hours, the Optical Density (OD) at 600nm was measured with a microplate reader when the growth control was good and the MIC value of the quality-controlled strain was within the range given in the CLSI M27-A2 protocol. The growth rate is the OD value/growth control OD of each well, and the minimum inhibitory concentration of each strain is judged by taking the inhibition of more than 80% of the growth of the fungus as a standard.
(2) Checkerboard microdilution assay
The minimum inhibitory concentrations of each drug were determined according to the checkerboard microdilution protocol CLSI M27-A2. This is determined by its minimum inhibitory concentrationThe range of drug concentrations used in the experiment was adjusted to 4-fold working concentration by diluting the drug with RPMI-1640 liquid medium. Absorbing 50 mul of azole drugs from high to low concentration, respectively adding into the 12 th to 2 nd columns of a 96-well plate, and adding 50 mul of liquid culture medium into the 1 st column; 50 mul of AgNPs liquid medicine is sucked from high to low concentration and respectively added into the A-G line and the H line of a 96-well plate, and 50 mul of liquid culture medium is correspondingly added into the H line. Then 0.5-2.5 multiplied by 10 of the adjusted concentration of the bacterial suspension3cfu/ml 100. mu.l was added to each well. H1 is a growth control without drug. This resulted in 77 combinations of the two drugs at different concentrations. The 96-well plate is placed in a 37 ℃ constant temperature incubator for 48 hours, and then the result is measured and recorded at 600nm by a microplate reader. All experiments were repeated three times. For the evaluation of bactericidal effect of the combination, 5. mu.l of each sample in a 96-well plate was spotted on YPD plates by a plate spotting method, and then they were cultured in a 37 ℃ incubator for 48 hours and then photographed to analyze their bactericidal effect.
(3) Evaluation and determination method
The nonparametric method of the LA model adopts Fractional Inhibition Concentration Index (FICI) with the formula of Sigma FIC (FIC ═ FIC)A+FICB(MIC a, combination/MIC a, used alone) + (MIC B, combination/MIC B used alone).
Determining the FIC index: calculating all FIC values in the data table, wherein when all the FIC values in the data table are less than 4, the value with the minimum FIC is the FIC value; otherwise, the maximum value of the FIC value is FIC. FIC is less than or equal to 0.5, and shows a synergistic effect; FIC >4 is antagonistic; between the two is irrelevant. A smaller FIC index indicates a stronger synergy between the two drugs [ OddsFC.2003.synergy, antadonisms, and what the Chequerboard puts between the m.J. AntichronobChromo. 52:1 ].
An isobologram solution method: the minimum inhibitory concentration of each drug was plotted by isobologram by chessboard dilution, and the two drugs were considered to have synergistic effect if the MIC values of the two drugs were reduced to 1/4 of the MIC values of the single drug, which means that the sum of FICs of the combined drugs was equal to or less than 0.5. Synergy (convex), antagonism (concave) and irrelevance (straight line) [ Te dorso DTA, Verweij PE, Meletiadis J, Bergervoet M, Punt NC, MeissFGM, motion JW.2002.in the visual interaction of flucytosed composite with an amphotericin B or flucytosed imaging third-five layer analytes determined by the reactive of the positive ions, and the reactive surface of the negative ions chemistry 2989] can be judged from the shape of the curve.
(4) Results of sensitivity analysis and checkerboard microdilution (see Table 1)
The results of the sensitivity analysis, see table 1, show: the MIC value range of AgNPs to 10 clinically isolated Candida albicans is 16-32mg/L, and no obvious difference exists between azole sensitive bacteria and azole drug-resistant bacteria.
The results of the checkerboard microdilution method, see table 1, show: for drug-resistant strains CA10, CA135 and CA137, the MIC of the drug can be obviously reduced by combined administration, for example, for CA10, the MIC value of fluconazole is reduced from 256mg/L to 1mg/L after combined administration; the MIC value of voriconazole is reduced from 64mg/L to 0.25mg/L, and the MIC value can be reduced by more than 200 times. The in vitro action relationship of the compounds is a synergistic action relationship to the drug-resistant strains; and the synergistic or unrelated action relationship is realized on sensitive strains.
TABLE 1 checkerboard microdilution assay for Candida albicans and FICI evaluation of AgNPs and azole drugs against 10 clinical isolates
Wherein,
aMIC is the minimum inhibitory concentration;
bFICI is graded bacteriostatic concentration index;
cSYN stands for synergy; ANT represents antagonism; IND represents no correlation. And (3) synergy: FICI value is less than or equal to 0.5, antagonism: FICI value>4.0, and an irrelevant FICI value of 0.5-4.
dFLC, fluconazole;
eVOR, voriconazole.
The growth rate data for fluconazole in combination with AgNPs against drug-resistant candida albicans CA10 is shown in table 2. The concentration ranges of AgNPs and fluconazole are 0-8mg/L, and 77 drug combinations with different concentrations are arranged in the middle.
The minimum value of 0.02 and less than 0.5 is obtained by calculating the corresponding drug concentration of the growth rate of less than 20 percent and calculating the FICI value, which indicates that the AgNPs and the fluconazole are in a synergistic action relationship when being applied together.
TABLE 2 FICI index analysis AgNPs and Fluconazole combination anti-azole-resistant Strain CA10
FLC, fluconazole; FICI, graded inhibitory concentration index.
In FIG. 2, the A diagram shows the equivalence analysis of the drug-resistant Candida albicans CA10 for the combination of fluconazole and AgNPs, and the equivalence curve is concave, which shows that the combination of the fluconazole and the AgNPs is in a synergistic relationship.
The panel B in FIG. 2 shows the bactericidal effect after the plate is dripped, and the result shows that the two are combined to have a bactericidal synergistic effect.
(5) Agar diffusion assay (Agar diffusion assay): selecting a clinical drug-resistant strain CA10, inoculating the strain into 10ml YPD liquid culture medium, culturing at 37 ℃ and 200rpm overnight with shaking, and then activating the bacterial liquid again. 1ml of the inoculum was centrifuged, resuspended in physiological saline, counted on a hemocytometer, diluted with physiological saline and adjusted to 5X 107 CFU/ml. 100 μ l of the bacterial suspension was added to 10ml of preheated YPD medium containing 0.75% agar, mixed well and poured quickly into YPD solid petri dishes prepared in advance, on the surface of which were placed sterile filter paper discs (diameter 6mm) containing different doses of the drug, and blank controls containing the corresponding equivalent amount of solvent. The agar culture dish was cultured in an incubator at 37 ℃ for 48 hours, and then the state of the zone of inhibition was observed.
The experimental result of the agar diffusion method is as follows:
the graph C in FIG. 2 shows the results of the experiments performed by the agar diffusion method. For drug-resistant bacteria CA10, azole drugs fluconazole 10 μ g, voriconazole 4 μ g have no obvious bacteriostatic action on YPD, and the antibacterial action of 2 μ g AgNPs is also weaker, but the size of a bacteriostatic zone can be obviously increased by combined administration, and the bacteriostatic zone can grow aseptically.
(6) Time-sterilization curve (Time-kill test)
The clinical drug-resistant candida albicans CA10 is activated twice in YPD culture solution, and is subjected to shaking culture at 37 ℃ and 200rpm to enable the fungus to be in the later exponential growth phase. The bacterial solution was taken to 1ml, centrifuged and resuspended in physiological saline, and the cells counted and adjusted to a bacterial density of 108 cfu/ml. The bacterial liquid is diluted to 104cfu/ml by RPMI-1640, and divided into 4 parts, each part is divided into 20ml, and corresponding medicines are respectively added to the final concentration: the concentration of PVP-AgNPs is 4mg/L, and the concentration of fluconazole and voriconazole is 1 mg/L; the blank was filled with the same volume of DMSO, and the DMSO content in all the bacterial solutions was less than 1%. Then incubated at 37 ℃ and measured after 0, 12, 24, 48 and 72 hours, respectively.
The determination method comprises the following steps: viable bacteria counting method: 100. mu.l of each tube of the culture broth was serially diluted 10-fold with 0.9% physiological saline, and 50. mu.l of each tube of the culture broth was uniformly applied to the surface of YPD solid medium. The number of colonies was counted after culturing the plates in a 37 ℃ fungal incubator for 48 hours, with the number of colonies growing on YPD plates reaching 20 as the minimum. Viable colony Counts (CFU) were calculated and plotted against time at Log10 CFU/ml. The correlation between the two was evaluated.
The effect evaluation method of the combined medication comprises the following steps: compared with the single antifungal drug with the highest activity, the synergistic effect of the two drugs is confirmed when the bacterial concentration reduction value is more than or equal to 2Log10CFU/ml after the two drugs are used together; when the decrease in the bacterial concentration after the combination of the two drugs is less than or equal to 2Log10CFU/ML, compared with the case where the Antifungal agent alone is most active, it is confirmed that the two drugs have an independent effect [ Haynes MP, Buckley HR, Higgins ML, Pieriger RA.1994.Synergism between the antibiotic AgentsAmphotericin B and Alkyl Glycerol ethers. antibiotic Agents Chemothers.38: 1523. 1529 ].
The sterilization curve experiment result is as follows:
the graph D in fig. 2 shows the results of the experiments verified by the method using the time-sterilization curve. After 72 hours, the number of cells was 8.4LogCFU/mL for Candida albicans CA10 in the control group, 8.23LogCFU/mL for CA10 in the PVP-AgNPs single treatment group, 6.8LogCFU/mL for CA10 in the fluconazole single treatment group, and 4.56LogCFU/mL for CA10 in the PVP-AgNPs-fluconazole combination treatment group. Compared with single drug, the LogCFU/mL bacterial count is reduced by more than 2LogCFU/mL after the fluconazole and the PVP-AgNPs are jointly applied, and the synergistic effect of the combined drug is verified. After 72 hours, the cell number of the single-use drug group of the voriconazole, namely candida albicans CA10, is 7.1LogCFU/mL, the cell number of the LogCFU/mL is reduced by 2.2LogCFU/mL after the voriconazole and PVP-AgNPs are combined, and the synergistic effect of the combined application of the voriconazole and the PVP-AgNPs is verified.
Example 2 study of cytological Co-operative mechanisms
(1) The method for researching the adhesion of AgNPs to the fungal cells comprises the following steps: 1X 107CFU/mL Candida albicans CA10 was treated with the drug for 24 hours, observed under a 100-fold oil mirror, and photographed. Statistics were analyzed using ImageJ software, with at least 100 fungal cells analyzed per sample.
The results show that: the addition of fluconazole increased the adherence of AgNPs to fungal cells and the ratio of adherence increased with increasing concentration (fig. 3A and 3B), and the combination significantly reduced the germination rate of fungal cells (fig. 3C).
(2) Propidium Iodide (PI) absorption analysis study method: propidium Iodide (PI) is used as an indicator probe of the increase of the permeability of the fungal cell membrane, PI can not pass through a live cell membrane but can pass through a damaged cell membrane to stain a nucleus, and Candida albicans after drug treatment is cultured with 5mg/L PI37 ℃ in the dark for 15 minutes and is rinsed with PBS 3 times to be observed and photographed under a fluorescence microscope.
The results show that: the combined drug can obviously increase the red fluorescence quantity of PI, and the permeability of the fungal cell membrane is increased after the combined drug is used (figure 4A and 4B).
Example 3 molecular mechanism study
(1) Method for measuring expression of related genes in ergosterol biosynthesis pathway (ERG3, ERG5, ERG1, ERG25, ERG6 and ERG11) and fungal cell membrane efflux pump related genes (MDR1, CDR1 and CDR2) by fluorescent quantitative PCR (polymerase chain reaction) 1.1 sample treatment
Selecting drug-resistant strain CA10, inoculating to 30ml YPD culture solution, culturing at 37 deg.C overnight, centrifuging at 3000g for 5min, and resuspending with fresh YPD to adjust bacterial density to 1.0 × 106cfu/ml of drug group and control group. Shake-culturing at 37 ℃ for 12 hours, and centrifuging at 4 ℃.
1.2TES preparation
10mM Tris.Cl pH7.5; 10mM EDTA; 0.5% (W/V) SDS was prepared in RNase-free water and stored at room temperature.
1.3 extraction of RNA by thermal phenol method
The pellet was resuspended in 1ml ice-cold PBS and transferred to 1.5ml proteinase K-treated Eppendorf tubes, 3000g and centrifuged for 10 min. The supernatant was discarded, the pellet was suspended in 400. mu.l TES solution, 400. mu.l acidic phenol was added, and the mixture was vigorously shaken for 10 seconds with a vortex shaker. Transferring to 65 deg.C water bath, incubating for 30-60min, and shaking vigorously with vortex oscillator for 10s every 5 min. Taking out, ice-cooling for 5min, and centrifuging at 4 deg.C at 10000g for 5 min. The upper aqueous phase was transferred to a 1.5ml proteinase K-treated Eppendorf tube and chloroform was added to precipitate the protein. Shaking vigorously, and centrifuging at 4 deg.C at 10000g for 5 min. The supernatant was transferred to a 1.5ml proteinase K-treated Eppendorf tube and added to 2/3 volumes of isopropanol and allowed to stand at 4 ℃ for 2 h. Centrifuging at 4 deg.C 10000g for 8 min. The supernatant was discarded, the pellet was suspended in ice-cold 70% ethanol, and the RNA pellet was washed with rapid shaking. Centrifugation at 10000g for 8min at 4 ℃ removed the supernatant as thoroughly as possible, dried at room temperature and the precipitate dissolved in 15. mu.l of RNase-free water. The samples were assayed for absorbance at 260nm and 280nm, respectively, the purity of the RNA was determined and the mass of RNA was calculated and stored at-80 ℃. Contamination with rnases is strictly prevented throughout the RNA extraction process.
1.4 Synthesis of cDNA by reverse transcription
Reverse transcription was performed according to the reverse transcription kit instructions to reverse transcribe total RNA into cDNA. The method comprises the following steps:
1) 2 mu g of extracted total fungal RNA is taken, 1 mu l of oligo (dT) is added, and the volume is increased to 12 mu l by adding RNase-free deionized water. And (4) centrifuging for 3-5 sec after uniformly mixing.
2) Denaturation at 70 ℃ for 5min, cooling on ice for 30sec, and centrifuging for 3-5 sec.
3) The reaction mixture was ice-cooled and the following components were added: 5 Xbuffer 4 μ L, RNase inhibitor (20U/μ L)1 μ L, dNTPs (10mmol/L)2 μ L, mixing, and centrifuging for 3-5 sec.
4) After a water bath at 37 ℃ for 5 minutes, 1. mu.l of reverse transcriptase (20U/. mu.l) was added in a total volume of 20. mu.l.
5) The reaction mixture was reacted at 37 ℃ for 60min, inactivated at 70 ℃ for 10min, quenched on ice and stored at-20 ℃ until use.
Fluorescent quantitative PCR
PCR was performed using cDNA as a template and 18S rRNA gene as an internal reference. The primer sequences used are shown in Table 3 below.
TABLE 3 qPCR specific primers
Reaction system:
amplification conditions: pre-denaturation at 95 ℃ for 5 min;
denaturation at 95 ℃ for 1min, annealing at 60 ℃ for 1min, and extension at 72 ℃ for 45s for 35 cycles.
Further extension was carried out at 72 ℃ for 5 min.
Negative control: PCR amplification was performed without the addition of cDNA template.
The results show that: the PVP-AgNPs and fluconazole combined drug can obviously up-regulate ERG5 and ERG6 genes and down-regulate CDR1 genes.
(2) Determination method and results of ergosterol:
the ergosterol is determined by a little improvement on the basis of the literature [ Prasad T, Chandra A, Mukhopadhyay CK, Prasad R.2006. unknown link between enzymes and drug resistance of Candida spp.: electron deletion of membrane flow and drug difference, and lead to drug-sensitive cells. antibiotic reagents Chemothers.50: 3597-:
selecting drug-resistant strain CA10 monoclonal strain, inoculating in 30ml YPD culture solution, culturing at 37 deg.CAt night, 3000g are centrifuged for 5min, and the cell density is adjusted by resuspension with fresh YPD at 1.0X 106cfu/ml of drug group and control group. Culturing at 37 deg.C with shaking for 12 hr, collecting cells, weighing wet bacteria weight, and extracting sterol. Adding 2.5ml PBS and 6ml freshly prepared saponifier (90% ethanol solution containing 15% sodium hydroxide), mixing, and saponifying in 80 deg.C water bath for 60 min. Extracting with 6ml petroleum ether (boiling range of 30-60 deg.C) for 2 times, mixing extractive solutions, washing with 6ml distilled water for 1 time, volatilizing ether layer under nitrogen to obtain unsaponifiable lipid, dissolving with cyclohexane to obtain 1ml/g wet bacteria, and storing at-20 deg.C.
Ultraviolet measurement: four characteristic peaks exist at the wavelength of 240nm-300 nm. Ergosterol and 24(28) -Dehydroergosterol (DHE) both have an absorption peak at 281.5nm, but only 24(28) -DHE has an absorption peak at 230 nm. The percent content of ergosterol can be determined by subtracting the absorption of 24(28) -DHE at 230nm from the total of 24(28) -DHE and ergosterol (by calculating the absorption at 281.5 nm). Namely by the equation:
ergosterol% +24(28) -DHE% ([ (a)281.5/290×F)]Wet weight of thallus;
24(28)-DHE％＝[(A230/518)×F]wet weight of thallus;
ergosterol%281.5/290×F)]Bacterial wet weight- [ (A)230/518)×F]Wet weight of thallus;
where F is the dilution factor and 290 and 518 are the E values (percent per cm), determined by the crystal structures of ergosterol and 24(28) -DHE. The experiment was repeated three times.
The results show that: for AgNPs alone, the treatment of Candida albicans resistant strain CA10 reduced the content of ergosterol by 25%, fluconazole caused the reduction of ergosterol by 86.2%, and the combined use of AgNPs and fluconazole (FIG. 6A) reduced the content of ergosterol significantly and by 92% compared to either drug alone (FIG. 6B).
Claims (4)
- The application of PVP modified nano silver and azole drugs in the preparation of drugs for resisting drug-resistant fungus activity combination.
- 2. The use of claim 1, wherein: the azole drug is selected from one or two of fluconazole and voriconazole.
- 3. The use of claim 1, wherein: the drug-resistant fungi is drug-resistant candida albicans.
- 4. A pharmaceutical composition characterized by: the PVP modified nano silver and azole drug are included, and the azole drug is selected from one or two of fluconazole and voriconazole.
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