CN110251497B - Application of robenidine hydrochloride in preparation of medicine for treating fungal infection - Google Patents

Abstract

The invention belongs to the field of medicine preparation, and particularly relates to application of robenidine hydrochloride in preparation of a medicine for treating fungal infection. Experiments prove that the minimum inhibitory concentration of the robenidine hydrochloride is 4 mu M, which is lower than that of the common antifungal drug fluconazole, the inhibitory effect of the robenidine hydrochloride is more obvious under the same concentration dose, and the robenidine hydrochloride still has good killing effect on fungus strains and super fungi which generate drug resistance, and has low toxicity, safety and reliability. The invention also discovers that the cell wall and the transcription factor RLM1 are the target of the robenidine hydrochloride for treating fungal infection, and provides a wide prospect for the drug treatment of fungal infection.

Description

Application of robenidine hydrochloride in preparation of medicine for treating fungal infection

Technical Field

The invention belongs to the field of medicine preparation, and particularly relates to application of robenidine hydrochloride in preparation of a medicine for treating fungal infection.

Background

Fungal infections have a tremendous impact on human health, with about a billion people worldwide each year suffering from fungal infections, resulting in over 150 million deaths. With the development of medical science, the use of various new drugs and new technologies such as broad-spectrum antibiotics, glucocorticoids, immunosuppressants, various catheter intubation technologies, organ and bone marrow transplantation is more and more common, and patients with immunodeficiency such as malignant tumors, HIV infection, the elderly population and the like are increasingly increased, so that the incidence rate and the mortality rate of opportunistic fungal infection caused by the immunodeficiency are increased year by year and are more and more valued by the medical field. The existing antifungal drugs can not meet the clinical requirements more and more, the research and development of novel drugs needs a long time, and the serious disconnection between the development speed and the social requirements is a difficult problem faced by the current medical field. On the other hand, the drug resistance problem of pathogenic fungi to current clinical antifungal drugs is more and more emphasized. These factors have increased the urgency for finding new antifungal drugs, have prompted the industry and research institutes to develop safer and more effective therapeutic drugs, discover new chemical entities, and ultimately treat pathogenic fungal infections. Therefore, the research and development of novel antifungal drugs and the search for new action targets are undoubtedly important hopes for solving the problems, and have important social and economic significance for human health science and the major health industry.

Candida albicans, the most common opportunistic pathogenic fungus, symbioses on skin and mucosal surfaces of healthy people without causing harm, but infects blood and tissues and organs to cause serious deep fungal infection when the host is immunodeficiency or low in immune function, and the death rate is up to 40% -80%. There are three major classes of drugs currently used clinically to treat fungal infections: polyenes, echinocandins and azoles, are specifically characterized as follows: 1) polyene antifungal drugs: among the most used are amphotericin B. The combination of the lipophilic structure of amphotericin B and ergosterol of fungal cell membrane increases the permeability of cell membrane, which results in leakage of important substances such as electrolyte and amino acid in cytoplasm and death of fungi. However, the drug has serious toxic and side effects (especially nephrotoxicity), and the fungal infection patient after the application of the drug to renal transplantation usually has the cost of damaging the renal function of the transplantation, and the drug dosage must be increased from a small dosage, so that the effective concentration cannot be rapidly reached, and the treatment risk is possibly increased, thereby being limited in clinical application. 2) Echinocandins: the main representatives are caspofungin and the like, the action mechanism of the caspofungin is to specifically inhibit beta (1,3) -D-glucan synthetase, interfere the synthesis of beta (1,3) -D-glucan of a fungal cell wall, cause abnormal cell wall structure, cell rupture and leakage of important contents, and finally cause the death of the fungal cell. Caspofungin is poorly absorbed orally, can only be administered intravenously and is expensive, with adverse reactions such as fever, local phlebitis, headache and histamine-like reactions. 3) Azoles: mainly comprises fluconazole and the like. The main action mechanism of the medicine is to selectively inhibit C14-alpha-demethylase dependent on fungal cytochrome P450, so that the accumulation of 14-methyl sterol in fungal cells and the synthesis of ergosterol are reduced, the permeability of cell membranes is increased, important substances in cells leak out, and the growth of the fungi is finally inhibited. Fluconazole is the most widely used first-line drug, but has bacteriostatic activity only, so that the occurrence rate of drug-resistant strains is greatly increased, and adverse reactions such as nausea, vomiting, abdominal pain, rash and the like are accompanied.

The limitation of the existing antifungal drugs and the abuse of clinical antibiotics and other problems cause the frequent appearance of drug-resistant strains, wherein the formation of a Biofilm (Biofilm) is an important reason for the generation of the drug resistance of candida albicans. Biofilms are complex structures formed by the accumulation of bacteria or fungi and their extracellular secretions on biological or non-biological surfaces, the formation of which mainly involves the stages of adhesion, initiation, maturation, detachment and diffusion, and after biofilm formation, they enhance the ability of microorganisms to defend against host immune attack and antibacterial drugs, and some pathogenic bacteria can escape from biofilms for transmission and diffusion, causing invasive infections. Candida albicans biofilm is structurally complex, consisting of various forms of cells (including yeast, hyphal and pseudohyphal cells) and extracellular matrix. The extracellular matrix can effectively protect the Candida albicans from attacking host immune cells, isolate the killing of antibacterial drugs, and provide a stable and rich nutritional environment for the growth of the Candida albicans. A drug resistance test research on a formed biomembrane candida albicans continuous perfusion antifungal drug shows that the candida albicans has high tolerance to fluconazole, and amphotericin B can effectively inhibit the diffusion of the candida albicans biomembrane only at high concentration; another strain test result obtained by clinical separation shows that 65.5 percent of clinical samples can be separated into Candida albicans, 15.1 percent of the Candida albicans strains obtained clinically have drug resistance to fluconazole and 24.7 percent of the Candida albicans strains have drug resistance to itraconazole. Thus, biofilm formation has become a problem in combating fungal infections. In order to solve the problem, in addition to the research and development of new drugs, one of the new antifungal strategies is to discover the antifungal effect of the existing drugs and achieve the synergistic effect in resisting fungal infection by combining with the clinically common antifungal drugs.

Fungal cell walls are highly dynamic complex structures that are critical for maintaining cell shape and protecting the environment. The firmness of the cell wall is critical to maintaining the morphology of the fungus. Mutations that disrupt the molecular integrity of the cell wall result in loss of the spatial morphology of the ovular, pseudohyphal, and hyphal cells, often leading to lysis and death. The cell wall is also the first point of contact between the fungus and the host, and therefore the cell wall is critical for fungal-host interaction and immune recognition. The cell wall consists of an inner framework layer of chitin and beta-glucans ( beta 1,3 and beta 1, 6-glucans) and an outer layer of hyperglycosylated mannoproteins. These proteins are modified with linear O-linked mannans and highly branched N-linked mannans, which can be linked with additional mannan side chains through phosphodiester linkages known as Phosphomannans (PMs). The mannan portion of the cell wall is important for adhesion, cell wall integrity, immune recognition, and comprises up to 40% of the dry weight of the cell wall. Thus, many of the features of fungal cell wall biosynthesis are characteristic of fungi and are therefore considered to be good targets for the development of antifungal drugs.

Chlorobenzidine hydrochloride is widely used in chicken and rabbit feeding as an anti-coccidiosis veterinary drug, and researches show that the residual dosage of the drug in edible tissues is reliable and safe to human bodies and has no toxic or side effect, and no research result of the drug on the fungus inhibition effect is published at present.

Disclosure of Invention

The invention aims to provide application of robenidine hydrochloride in preparation of a medicine for treating fungal infection. Experiments show that: the robenidine hydrochloride has obvious bacteriostatic effect, has good killing effect on fungus strains and super fungi which generate drug resistance, and has low toxicity, safety and reliability.

The technical scheme provided by the invention is to provide the application of robenidine hydrochloride in the preparation of the medicine for treating fungal infection. The fungi comprise strains of Candida, Cryptococcus, Saccharomyces, and Aspergillus.

Further preferably, the strain of candida is candida albicans; the strain of cryptococcus is cryptococcus neoformans; the strain of Saccharomyces cerevisiae is Saccharomyces cerevisiae; the strain of Aspergillus is Aspergillus fumigatus.

The fungi include drug-resistant fungi and super fungi.

More preferably, the drug-resistant fungus is candida albicans drug-resistant strain; the super fungus is Candida auriculata.

A medicine for treating fungal infection, which is characterized in that the medicine is a substance for treating through taking a transcription factor RLM1 as a medicine target. Preferably, the fungus is candida albicans.

A medicine for treating fungal infection is characterized in that the medicine is a substance which is treated by taking fungal cell walls as medicine targets. Preferably, the fungus is candida albicans.

More preferably, the active ingredient of the substance contains robenidine hydrochloride.

References to the transcription factor RLM1 are: bruno VM, et al (2006) Control of the C.albicans cell wall map response by mapping regulator case 5.PLoS Patholog 2(3): e21

DNA source：

ATGGGTAGAAGAAAGATTGAAATAGAACCATTGACAGACGATAGAAATCGTACAGTGACTTTTGTGAAGCGTAAGGCAGGGTTATTTAAAAAAGCTCATGAATTAGCTGTGCTCTGTCAAGTGGATTTAACGGTTATTATCGTTGGCAATAATAATAAAGTATATGAATATTCTACTGTTGAGGCAAATGAGATTTTTAATGCCTATAATAAAACCATTAAAGTCAGAAAACAAGTACATGAATCGAAGTCTCCAGAATATTATTCGAAATTTAGAAAGAAACGACATTTAAATGAACCACTTATGAATAAATCAGGGTCTGTAGTTGGCACTAATACACATTTGAACGATGAAGACTATGATCATAATGTTCATGAAGCGGGCGATGAGGATTCGGAATATGAAAGCGATGATAATTCTCCACAACCTAAACGGCACAAAAGATCAGAGTCGGTTAAAAAAGAGCAAAACCCCAAAGTGTTTAATAGTACCCAACCTCCACCACCGCCTCCACCACCTCATATATCTTTAAATAATGTTCCAACATTTACCAACCCCCAAAATTACAAAAAACAGATTGATGAGACAAATAACACTTCGGCACCGCCCGCTACTGGGACAAAAAATGAACCAACGATGCAACGACCAGTATTGAGGGTACAAATACCGAATGATGCCAAGAGCAATACGAATAATTCCCATAGTGGTGTTAATAATAGTGATGGCAAGGACACGGCGAGAACAGTGACGGCAGTCGACAATAGTGCAACCAACCAAAACACTCAATCGAGCAATACAACATCAGGTACAGGGACTGCTGATACCAATTCATCGCAACTAAATTCAAATGGTAATAGTAATTTAGTGCCTGCGAATGTTCCAAATACCAGGTTTTCGGGATATTCATCGTTTCGATCACCAGACTCACGAAAACCAACACTACCGTTACCTTTGCAAACCAAATCACAAACGTCATCTCCAGCTAGTGCTGTAGCACCAGGTTTACCATTGACAGGAGGAAGCAATGCATATTTTGCAGGAATGCAACAATCACCCGTGGGTGGTTCGTATGTCAATTATCCAGCCCAAGTATATCAGCAGTATCAACAGTTCCAAAATCAACTACAACTACAAGAACAACAACAGCAACAGCAAAAACAACAATCTCAGCCGCAGCCATCATCGCAACTGGTTGGAAATCAAAATGCACAATTGGAATCAGCAGCACGATTCCGTTCTGGTTTACCGACAGGGACACAATTTAATAATGGTGAACAAACACCAATTTCAGGATTGCCATCACGATACGTTAATGATATGTTCCCCTTCCCATCTCCATCAAACTTTCTTGCACCTCAAGATTGGCCATCAGGTATAACACCAACTACTCATCTACCACAGTATTTTGTGAATATGCCATTGAGTGGAATTGGACTGCAACAACTGCAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAGCAACAACAGCAACAACAGCAGCAGAATGCCCAACAGCAACTGCAAGTACCTGTTATCCCAATACAAACACAAACATCACAACAAATGGCTTCAACTACCAATCACAAATCAGCTAATCTAATACCAGGGTTTTTACAAAACCCAACACAAGCCACTGGAAATTCGGCAAATGCTTCCAAGCTGAGTGATGCTGGTGATGGTACTAATCCAACCACAGCAGGAAGTTCAAGTTCAGCAGATGTCAATAACACCAACAATGGACCTAATAAAAATACATAA

The invention has the beneficial effects that:

1. the robenidine hydrochloride has obvious bacteriostatic effect, the minimum bacteriostatic concentration of the robenidine hydrochloride is 4 mu M, which is lower than that of the common antifungal drug fluconazole, and the bacteriostatic effect of the robenidine hydrochloride is more obvious under the same concentration dose.

2. The robenidine hydrochloride still has good killing effect on fungus strains generating drug resistance, so that a potential solution is provided for the current situation of clinical ubiquitous fungus drug resistance.

3. The robenidine hydrochloride has the same good bacteriostatic effect on candida auriculata, and has certain reference significance for treating diseases caused by 'super fungi' in the future.

4. The invention discovers that the cell wall and the transcription factor RLM1 are the target of the medicine for treating fungal infection by using robenidine hydrochloride.

5. The chlorbenzoguandine hydrochloride has cytotoxicity only when the concentration is 64 mu M, and the animal fed with the chlorbenzoguandine hydrochloride has low drug concentration in muscle tissues which is far less than the dosage harmful to human health, so the chlorbenzoguandine hydrochloride has low toxicity, safety and reliability.

Drawings

FIG. 1 is a graph showing the growth of Candida albicans treated with fluconazole and robenidine hydrochloride, respectively, in example 1. Wherein, A is the dose-effect relation of fluconazole in YPD medium; graph B shows the dose-response relationship of robenidine hydrochloride in YPD medium.

FIG. 2 is a graph comparing the bacteriostatic effects of proguanil hydrochloride on clinical fluconazole-resistant strains and Candida albicans in example 2, wherein A is a dose-effect comparison of fluconazole and proguanil hydrochloride on Candida albicans in YPD medium; panel B is a dose-effect comparison of fluconazole and proguanil hydrochloride on drug-resistant strain #16 in YPD medium; panel C is a dose-effect comparison of fluconazole and proguanil hydrochloride on drug-resistant bacterium #17 in YPD medium; graph D compares the dose-effect of fluconazole and robenidine hydrochloride on candida auriculata in YPD medium.

FIG. 3 is a graph comparing the bacteriostatic effect of robenidine hydrochloride on other pathogenic fungi in example 3. Wherein, A picture is the bacteriostasis of the robenidine hydrochloride on the candida albicans; b, the bacteriostasis of the robenidine hydrochloride to the saccharomyces cerevisiae; the C picture shows the bacteriostasis of the robenidine hydrochloride on the cryptococcus neoformans; and the D picture shows the bacteriostasis of the robenidine hydrochloride on the aspergillus fumigatus.

FIG. 4 is a graph comparing the effect of robenidine hydrochloride on the production of Candida albicans hyphae in different media from example 4. Wherein, A picture shows the hyphal growth of Candida albicans in the Spider culture medium; panel B shows hyphal growth of Candida albicans in M199 medium; panel C shows the hyphal growth of Candida albicans in YPD plus 10% bovine serum medium.

FIG. 5 is a graph showing that robenidine hydrochloride inhibits the growth of Candida albicans biofilm in example 5. A is a comparison graph of photographing results of biofilm formation; the B picture is a comparison picture of quantitative analysis pictures of the biological membrane formed by the A picture.

FIG. 6 is a graph showing the effect of the robenidine hydrochloride drug on the cell wall of Candida albicans in example 6, wherein graph A is a graph comparing the stress response of Candida albicans cells to robenidine hydrochloride under different environmental pressures; b is a comparison graph of cell wall integrity determined by an alcian blue staining method.

FIG. 7 is a comparative photograph of a TEM image of the cell wall of Candida albicans treated with robenidine hydrochloride in example 6.

FIG. 8 is a graph comparing the effect of robenidine hydrochloride on different cell wall components in example 6. A is a graph of fluorescein isothiocyanate staining results of cell wall mannan; panel B shows CFW (calcium fluorescent white) fluorescent staining experiment of cell wall chitin; and the C picture is the quantitative analysis picture of the WGA-lectin (wheat agglutinin) fluorescence staining experiment of the chitin exposed on the cell wall surface.

FIG. 9 is a graph of activation of cell wall repair pathway in example 6 as determined by Western blot of crude protein extract of exponentially growing cells.

FIG. 10 is an analysis chart of the RNA sequencing results of Candida albicans treated with robenidine hydrochloride in example 6.

FIG. 11 is a graph showing cytotoxicity test of robenidine hydrochloride against FaDu epithelial cells and RAW-BLUE macrophages by Lactate Dehydrogenase (LDH) release in example 7.

Detailed Description

The invention will now be further illustrated by reference to the following examples:

the robenidine hydrochloride used in the experiments of the present invention was purchased from Targetmol.

Example 1

The growth conditions of Candida albicans treated with fluconazole and robenidine hydrochloride respectively.

Inoculating a small piece of the strain from the cryopreserved Candida albicans strain with a sterile inoculating loop into YPD-containing solid medium, and incubating overnight (15 hours) in an incubator at 30 ℃; after overnight incubation of the strain, it was washed twice with sterile PBS; measuring the concentration of the bacterial liquid with enzyme-labeling instrument, and adding YPD culture medium to make it OD₆₀₀0.4; respectively dissolving robenidine hydrochloride and fluconazole dissolved in DMSO (dimethyl sulfoxide) in a fresh YPD culture medium to obtain required concentrations, diluting with a 96-well plate by 2 times, adding 100 μ l of the prepared culture medium containing bacterial liquid, and supplementing 200 μ l of each well to obtain OD₆₀₀The OD is measured after shaking up 1 muM, 2 muM, 4 muM, 8 muM, 16 muM and 32 muM from low to high in the order of 0.2₆₀₀And measured every hour up to 24 hours. As shown in particular in figure 1. Graph A shows the dose-effect relationship of fluconazole in YPD medium; graph B shows the dose-response relationship of robenidine hydrochloride in YPD medium. (SC5314, Candida albicans, Flu for fluconazole, Robe for robenidine hydrochloride, all of which are substituted).

Absorbance OD of Candida albicans bacterial liquid₆₀₀As an indicator, the growth of the cells was monitoredAnd (4) concentration. After the growth curves of the candida albicans treated by the robenidine hydrochloride and the fluconazole respectively are analyzed, the robenidine hydrochloride has more obvious bacteriostatic effect compared with a control group after the strain is cultured in an YPD culture medium for 24 hours at the constant temperature of 30 ℃, and the growth curve is horizontal curve, OD₆₀₀The values were unchanged for 24 hours and completely inhibited the growth of Candida albicans at a concentration of 16. mu.M, as can be seen from the figure, the MIC (minimum inhibitory concentration) of robenidine hydrochloride was 4. mu.M, which is less than that of the positive control fluconazole. The experiments show that under the same dosage, the robenidine hydrochloride has better effect of inhibiting the growth of candida albicans compared with fluconazole.

Example 2

Bacteriostatic effect condition of robenidine hydrochloride on clinical fluconazole drug-resistant strains and candida auriculata

The experimental procedure was the same as in example 1, except that the test strains were replaced with drug-resistant strains and Candida auricular.

As shown in fig. 2, compared with the fluconazole control group, the robenidine hydrochloride has significant bacteriostatic action on two drug-resistant bacteria #16 and #17 (candida albicans drug-resistant strains extracted from tumor patients in clinical treatment), and in cells treated at different concentrations, the robenidine hydrochloride has a dose-dependent effect and gradually enhances the inhibitory action on the cells from low to high. In addition, candida auricula is also added in the experiment, and can be obtained from D in figure 2, the robenidine hydrochloride also has a strong inhibition effect on candida auricula, and the robenidine hydrochloride has a good clinical significance for treating super fungal infection.

FIG. 2A is a dose-response comparison of fluconazole and robenidine hydrochloride on Candida albicans in YPD medium; FIG. 2B is a comparison of dose-effect of fluconazole and robenidine hydrochloride on drug-resistant strain #16 in YPD medium; FIG. 2C is a comparison of dose-effect of fluconazole and robenidine hydrochloride on drug-resistant strain #17 in YPD medium; FIG. 2D is a dose-effect comparison of fluconazole and robenidine hydrochloride on Candida auriculata in YPD medium.

Example 3

The robenidine hydrochloride has obvious bacteriostatic effect on common human pathogenic fungi such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Cryptococcus neoformans (Cryptococcus neoformans) and Aspergillus fumigatus (Aspergillus fumigatus).

The experimental procedure is the same as that of example 1 except that the test strains are replaced by Saccharomyces cerevisiae, Cryptococcus neoformans and Aspergillus fumigatus.

The experimental results are shown in fig. 3, the growth curves of the saccharomyces cerevisiae and the candida albicans are very similar, and the minimum inhibitory concentration is 4 mu M; the aspergillus fumigatus and the cryptococcus neoformans have obvious dose-effect relationship, and can completely inhibit the growth of candida albicans at the concentration of 32 mu M (the growth curve is in a horizontal state within 48 hours). The experiments show that the robenidine hydrochloride has good inhibition effect on the clinical common pathogenic fungi.

FIG. 3A shows the bacteriostatic effect of robenidine hydrochloride on Candida albicans; FIG. 3B shows the bacteriostatic action of robenidine hydrochloride on Saccharomyces cerevisiae; FIG. 3C is a graph showing the bacteriostatic effect of robenidine hydrochloride on Cryptococcus neoformans; FIG. 3D shows the bacteriostatic effect of robenidine hydrochloride on Aspergillus fumigatus.

Example 4

Effect of robenidine hydrochloride on hyphal formation.

Candida albicans has three forms, i.e., yeast type, pseudomycelial type, and mycelial type, in which hyphal formation is critical to the pathogenesis of Candida albicans. To examine the effect of robenidine hydrochloride on hyphal production, we induced hyphae in different media.

1) Taking a small strain out of the frozen strain by using a sterile inoculating loop, inoculating the small strain into a YPD-containing solid culture medium, and incubating overnight (15 hours) in a constant temperature box at 30 ℃;

2) after overnight incubation of the strain, it was washed twice with sterile PBS;

3) measuring the concentration of the bacterial liquid with enzyme-labeling instrument, and adding YPD + 10% bovine serum culture medium, M199 culture medium, and Spider culture medium to make OD₆₀₀0.001. And setting a DMSO control group, a 4 mu M Robenidine group and an 8 mu M Robenidine group, and respectively adding DMSO with corresponding concentration and prepared Robenidine liquid medicine. Mixing, culturing in 37 deg.C incubator for 2 hr and 4 hr, observing with microscope, and taking picturesAnd the objective multiple is 40.

Experiments show that after 2 hours of culture in Spider and M199 culture media at 37 ℃, Candida albicans in the drug-added group exists in a yeast type morphology, no hypha generation is observed, and the hypha growth of a control group is obvious; in YPD medium containing 10% bovine serum, hyphal formation was observed in the drug-added group, but as compared with the control group, the drug-added group was significantly shorter in hyphal length than the control group. The experimental results prove that the robenidine hydrochloride can inhibit the generation of candida albicans hyphae in different hypha induction culture media.

Initial concentration of cells OD₆₀₀The results of photographs taken after 2 hours of incubation at 37 ℃ in three media are shown in fig. 4. FIG. 4A shows the hyphal growth of Candida albicans in Spider medium; FIG. 4B shows hyphal growth of Candida albicans in M199 medium; FIG. 4C shows the hyphal growth of Candida albicans in YPD plus 10% bovine serum medium.

Example 5

The robenidine hydrochloride inhibits the formation of candida albicans biofilm.

1) Mu.l of RPMI-1640 medium containing Candida albicans was inoculated into 96-well plates in columns 1 to 11, 8 wells in column 12 as a blank without addition of the culture. Each drug is provided with a certain concentration gradient, and each concentration is provided with 3 technical repetitions;

2) covering a 96-well plate after inoculation, sealing the plate with a sealing film, and placing the plate in an incubator at 37 ℃ for culturing for 24 hours;

3) after 24-48h, the biofilm with the three-dimensional structure is formed, and can be used for the subsequent antifungal drug susceptibility test. The specific incubation time is determined according to the objective of the study, if the study starts to perform adhesion culture for 4h

4) After the biofilm is formed, the culture solution is slightly sucked out;

5) wash 1 time with sterile PBS

6) The robenidine hydrochloride is prepared into a certain working concentration in the RPMI-1640 culture solution. Adding 200 mul of medicine working solution into the corresponding hole of the 1 st column of the 96-hole plate by using a multi-gun head pipette gun;

7) 100 μ l of RPMI was added to wells in columns 2 to 11, and no liquid was added in column 12 as a negative control;

8) sucking 100 mul of liquid from each hole in the 1 st row, adding the liquid into the corresponding hole in the 2 nd row, and lightly blowing, beating and uniformly mixing;

9) sucking 100 mul of liquid from each hole in the 2 nd row, adding the liquid into the corresponding hole in the 3 rd row, and lightly blowing, beating and uniformly mixing;

10) repeating the above steps in sequence, adding to the 10 th column and terminating; (columns 11 and 12 serve as positive and negative controls, respectively, without any treatment)

11) Covering a 96-hole plate cover, coating with a preservative film, and incubating in an incubator at 37 ℃ for 24 hours;

12) according to the experimental design, multiple tubes containing 10ml of XTT solution were prepared, and 1. mu.l of menadione was added to each tube to give a final menadione concentration of 1. mu.M. (ii) a

13) Add 100 μ l of XTT solution containing menadione to each well of 96-well plate;

14) coating a 96-well plate with aluminum foil, and incubating at 37 ℃ for 2-3 h;

15) the plate is opened, and each hole of the 96-hole plate is orange and gradually changed with the concentration of the medicine. A new 96-well plate is prepared and 75-80. mu.l of the solution is pipetted from the original plate into the new plate using a multigun pipette. The new plate is placed in a microplate reader to read OD₄₉₂；

16) Calculating the metabolic activity inhibition degree of each hole biological membrane according to the absorbance value reading, and drawing by using Graphpad software;

the result of the biomembrane experiment is shown in fig. 5, as shown in fig. 5A, the chlorobenzene guanidine hydrochloride medicament gradually increases from 0 to 128 mu M from left to right, and the bacterial liquid becomes clear and the bacterial amount gradually decreases; FIG. 5B is a graph showing the quantitative analysis of the biofilm formed in FIG. A; biofilm formation gradually decreased from 32 μ M and at 64 μ M by 50%, demonstrating that robenidine hydrochloride can inhibit candida albicans biofilm formation.

Example 6

The mechanism of the chlorophenidine hydrochloride for inhibiting fungi is verified.

1) Candida albicans needs to be in symbiosis with hostStress from the host microenvironment is to be handled at all times, so the strain itself generates a corresponding stress response during long-term evolution. To investigate whether the robenidine hydrochloride affects the stress response of the candida albicans to the external pressure, thereby inhibiting the growth of the candida albicans. The experiment used a solid culture medium dot plate method. The strains on each solid medium were arranged in four columns and five rows, each strain containing two replicates and five dilution gradients, with Candida albicans concentration OD in the top row₆₀₀0.5, dilute down a 5-fold concentration gradient to OD₆₀₀0.5, 0.1, 0.02, 0.004 and 0.0008 respectively, and 3 mul of the bacterial liquid is dripped into each point. Photographs were taken after 24 hours of incubation at 30 ℃. The results show that under different stress conditions of 1M sodium chloride, 0.1% Sodium Dodecyl Sulfate (SDS), 0.7M calcium chloride, 200 mug/ml calcium fluorescent White (Calco Fluor White), 200 mug/ml Congo Red (Congo Red) and 10 mug Caffeine (Caffeine), the growth of Candida albicans of all the added groups ("+") is more sensitive to the robenidine hydrochloride compared with the control group, and particularly is most sensitive to a group of treatment related to the cell wall pressure, which indicates that the robenidine hydrochloride treatment damages the cell wall of the Candida albicans, thereby inhibiting the growth of the Candida albicans under different external pressure conditions, and suggests that the action mechanism of the robenidine hydrochloride is possibly related to the cell wall integrity signal.

FIG. 6 shows the effect of robenidine hydrochloride drug on the cell wall of Candida albicans, and graph A shows the stress response of Candida albicans cells to robenidine hydrochloride under different environmental pressures; b is the alcian blue staining method for cell wall integrity and data represents the average amount of dye bound. P < 0.0001.

2) We examined the microstructure of the cell wall before and after drug treatment by transmission electron microscopy. As shown in FIG. 7, the cell wall of the control group (DMSO-treated) was intact, the cell contour was smooth and uniform, and after 8 μ M chlorobenzeneguanidine hydrochloride treatment, the cell wall damage was reduced and the cell sap leaked into the gap. After the concentration is increased to 16 mu M, the chlorobenzene guanidine hydrochloride treated cells have the phenomena of cell wall atrophy and more cell sap leakage, and experiments show that chlorobenzene guanidine hydrochloride destroys the cell walls of candida albicans.

3) In order to detect specific components of the damaged cell wall, a series of experiments are carried out, wherein a CFW staining method for detecting the content of total chitin, an FITC staining method for detecting the content of total mannan of the cell wall and a WGA-lectin fluorescence staining method for detecting the content of exposed chitin are adopted. As can be seen from fig. 8A, the fluorescence intensity of the cells in the control group is much higher than that of the drug-treated group, and the difference is very significant and statistically significant by quantitative fluorescence analysis. In FIG. 8B, it can be seen that the cell wall was uniformly stained in the control group, a highlight signal appeared at the site where the parental and daughter cells combined, i.e., the cell wall chitin component, and the strength of the cell wall was weakened after the drug treatment, even at the site where the two cells were connected, the strength was not significant, so the total mannan was significantly reduced after the drug treatment. FIG. 8C shows the fluorescence staining of the treated cells with WGA-lectin staining, thereby detecting the chitin fraction exposed to the outside of the cells, p < 0.0001. The amount of exposed chitin was reduced by the drug-treated cells by quantitative analysis, and the difference was statistically significant. The results show that after the treatment with the drug, the chitin on the cell wall of the candida albicans is damaged, and the total amount of mannan is obviously reduced.

4) To further identify the downstream signaling pathway involved in the disruption of the cell wall by robenidine hydrochloride, we examined the Mkc1 kinase signaling pathway involved in the cell wall integrity of candida albicans. After 1 hour and 2 hours of treatment, the cells were collected for Western blot and detected by phosphorylated Mkc1 antibody using Tubulin as loading control. As shown in fig. 9, we found that there was a dose-effect relationship for the phosphorylation of Mkc1 in cells treated with different drug concentrations. When the concentration of robenidine hydrochloride is increased from 4 to 16. mu.M, Mkc1 phosphorylation shows a gradient increase with increasing drug concentration, the signal is strongest at 16. mu.M, and the signal after 2 hours of treatment is weaker than that of the 1 hour treatment group. This result shows that Mkc1 phosphorylation was significantly enhanced when the cell wall was disrupted by robenidine hydrochloride, activating the cell stress response, while Mkc1 phosphorylation was reduced with time. In FIG. 9, activation of the cell wall repair pathway was determined by Western blotting of crude protein extracts of exponentially growing cells. The phosphorylated p44/42 antibody detects phosphorylated (activated) MKC1 protein. Lane 1 is a control, the cells are treated with dimethyl sulfoxide, lane 2 with 4. mu.M of proguanil hydrochloride for one hour, lane 3 with 8. mu.M of proguanil hydrochloride for one hour, lane 4 with 16. mu.M of proguanil hydrochloride for one hour, lane 5 with 4. mu.M of proguanil hydrochloride for two hours, lane 6 with 8. mu.M of proguanil hydrochloride for two hours, lane 7 with 16. mu.M of proguanil hydrochloride for two hours, lane 8 is also a control, and the cells are treated with dimethyl sulfoxide.

5) FIG. 10 shows the result of RNA sequencing of Candida albicans treated with robenidine hydrochloride. RNA was extracted for sequencing analysis after 90 minutes of treatment with robenidine hydrochloride, and compared with the results of the DMSO control group.

In order to deeply research the gene expression profile of cells treated by robenidine hydrochloride, candida albicans was cultured in a liquid culture medium in a shaker at 30 ℃, cells treated by adding drugs and at different time points of a control group were finally collected, RNA was extracted, and data analysis was performed after library construction and sequencing, with the results shown in the table. The expression of a key transcription factor RLM1 is remarkably increased after 90 minutes, RLM1 plays an important role in Candida albicans hypha generation and biofilm formation, and RLM1 deletion can remarkably change the proportion of main components of a cell wall, such as increasing the content of chitin and reducing the content of glucan and mannan. As can be seen from the result of RNA sequence comparison of Candida albicans and the control group after 90 minutes of drug treatment (shown by an arrow), the expression of RLM1 in the drug treatment group is increased by about 8 times, which is consistent with the cell wall component detection results reported by other research results. This suggests that RLM1 is likely one of the important gene targets for drug action.

Example 7

Toxicity testing

The experiment takes endothelial cells FaDu and macrophages RAW-BLUE as model cells, and combines an LDH method to detect the activity of the cells treated by different concentrations. Lactate Dehydrogenase (LDH) release was used to detect the cytotoxicity of robenidine hydrochloride against FaDu epithelial cells and RAW-BLUE macrophages. After 24 hours of cell culture, the cells are incubated with robenidine hydrochlorideCultivation at 37 ℃ and 5% CO₂After incubation for 2 hours under the conditions, the release of LDH was detected according to the method described in the kit instructions, and OD was measured with a spectrophotometer₄₉₂The value is the toxicity value.

The results show that the drug has cytotoxicity only above 64 mu M, and the effective inhibitory concentration selected in the experiment is lower than 64 mu M, so the adopted concentration of the drug in the aspect of inhibiting the growth of the candida albicans is relatively safe.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention should not be limited by the disclosure of the preferred embodiments. Therefore, it is intended that all equivalents and modifications which do not depart from the spirit of the invention disclosed herein are deemed to be within the scope of the invention.

Sequence listing

<110> Shanghai college of medicine of transportation university

Application of chlorobenzeneguanidine hydrochloride in preparation of medicine for treating fungal infection

<141> 2019-07-24

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1806

<212> DNA

<213> RLM1

<400> 1

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tttgtgaagc gtaaggcagg gttatttaaa aaagctcatg aattagctgt gctctgtcaa 120

gtggatttaa cggttattat cgttggcaat aataataaag tatatgaata ttctactgtt 180

gaggcaaatg agatttttaa tgcctataat aaaaccatta aagtcagaaa acaagtacat 240

gaatcgaagt ctccagaata ttattcgaaa tttagaaaga aacgacattt aaatgaacca 300

cttatgaata aatcagggtc tgtagttggc actaatacac atttgaacga tgaagactat 360

gatcataatg ttcatgaagc gggcgatgag gattcggaat atgaaagcga tgataattct 420

ccacaaccta aacggcacaa aagatcagag tcggttaaaa aagagcaaaa ccccaaagtg 480

tttaatagta cccaacctcc accaccgcct ccaccacctc atatatcttt aaataatgtt 540

ccaacattta ccaaccccca aaattacaaa aaacagattg atgagacaaa taacacttcg 600

gcaccgcccg ctactgggac aaaaaatgaa ccaacgatgc aacgaccagt attgagggta 660

caaataccga atgatgccaa gagcaatacg aataattccc atagtggtgt taataatagt 720

gatggcaagg acacggcgag aacagtgacg gcagtcgaca atagtgcaac caaccaaaac 780

actcaatcga gcaatacaac atcaggtaca gggactgctg ataccaattc atcgcaacta 840

aattcaaatg gtaatagtaa tttagtgcct gcgaatgttc caaataccag gttttcggga 900

tattcatcgt ttcgatcacc agactcacga aaaccaacac taccgttacc tttgcaaacc 960

aaatcacaaa cgtcatctcc agctagtgct gtagcaccag gtttaccatt gacaggagga 1020

agcaatgcat attttgcagg aatgcaacaa tcacccgtgg gtggttcgta tgtcaattat 1080

ccagcccaag tatatcagca gtatcaacag ttccaaaatc aactacaact acaagaacaa 1140

caacagcaac agcaaaaaca acaatctcag ccgcagccat catcgcaact ggttggaaat 1200

caaaatgcac aattggaatc agcagcacga ttccgttctg gtttaccgac agggacacaa 1260

tttaataatg gtgaacaaac accaatttca ggattgccat cacgatacgt taatgatatg 1320

ttccccttcc catctccatc aaactttctt gcacctcaag attggccatc aggtataaca 1380

ccaactactc atctaccaca gtattttgtg aatatgccat tgagtggaat tggactgcaa 1440

caactgcaac aacaacaaca acaacaacaa caacaacaac aacaacaaca acaacaacaa 1500

caacaacaac aacaacaaca acaacagcaa caacagcaac aacagcagca gaatgcccaa 1560

cagcaactgc aagtacctgt tatcccaata caaacacaaa catcacaaca aatggcttca 1620

actaccaatc acaaatcagc taatctaata ccagggtttt tacaaaaccc aacacaagcc 1680

actggaaatt cggcaaatgc ttccaagctg agtgatgctg gtgatggtac taatccaacc 1740

acagcaggaa gttcaagttc agcagatgtc aataacacca acaatggacc taataaaaat 1800

acataa 1806

Claims (3)

1. The application of robenidine hydrochloride in the preparation of the medicine for treating fungal infection;

the fungi comprise strains of Candida, Cryptococcus, Saccharomyces, and Aspergillus;

the fungi comprise drug-resistant fungi and super fungi.

2. Use according to claim 1, characterized in that: the strain of candida is candida albicans; the strain of cryptococcus is cryptococcus neoformans; the strain of the saccharomyces is saccharomyces cerevisiae; the strain of Aspergillus is Aspergillus fumigatus.

3. Use according to claim 1, characterized in that: the drug-resistant fungi are candida albicans drug-resistant strains; the super fungus is Candida auriculata.

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