Application of Farnesol in regulating STING expression in vulvovaginal candidiasis

Abstract

The present invention discloses the use of Farnesol in regulating STING expression in vulvovaginal candidiasis. The present invention also provides the use of Farnesol in screening agents that promote the binding of Farnesol and STING, and in preparing STING expression regulators. The present invention provides various methods, including methods for screening drugs for treating vulvovaginal candidiasis, methods for screening compounds that affect the binding of Farnesol and STING, methods for regulating STING expression in in vitro cells, computer-assisted drug screening methods based on Farnesol and STING, and computer-assisted drug screening methods that promote the binding activity of Farnesol and STING. The present invention also provides a computer-assisted drug screening system based on Farnesol and STING, as well as a computer-assisted drug screening system that promotes the binding activity of Farnesol and STING. The present invention also provides computer-assisted drug screening equipment and computer-readable storage media.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of Farnesol in regulating STING expression in vulvovaginal candidiasis. Background Art

[0002] Vulvovaginal candidiasis (VVC) is a common vulvovaginal inflammation caused by the yeast Candida albicans. This pathogen is widely found in nature and also parasitizes resident fungi on human skin, vagina, mouth, and digestive tract. Yeast metabolites can irritate the vulva, causing severe itching, as well as the cervix and vagina, causing excessive fluid secretion. The multiplying bacteria also produce a protein coagulase that coagulates the protein components of vaginal discharge, resulting in a thick, curd-like discharge. Symptoms of this condition can sometimes include frequent and urgent urination. In those with weakened immune systems, it can lead to serious infections of the esophagus and other internal organs.

[0003] Farnesol is a naturally occurring 15-carbon organic compound and an acyclic sesquiterpenoid alcohol. Under standard conditions, it is a colorless liquid. It is hydrophobic and therefore insoluble in water, but miscible in oil. Farnesol is produced in both plants and animals from 5-carbon isoprenoid compounds. Phosphoric acid-activated derivatives of farnesol are likely the building blocks of all acyclic sesquiterpenoids. These compounds double to form 30-carbon squalene, a precursor to steroids in plants, animals, and fungi. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The present invention provides a pharmaceutical composition comprising Farnesol and a vector for overexpressing STING protein.

[0006] Furthermore, the vector includes a viral or non-viral vector.

[0007] Furthermore, the vector is capable of overexpressing STING protein.

[0008] Furthermore, the vector can be transduced into cells and expressed.

[0009] Furthermore, the expression refers to the transcription and translation of a nucleotide sequence into a protein / amino acid sequence.

[0010] Furthermore, the cells include vaginal epithelial cells.

[0011] Furthermore, the pathogen is vulvovaginal Candida.

[0012] Furthermore, the drug also includes a pharmaceutically acceptable adjuvant.

[0013] Furthermore, the pharmaceutically acceptable adjuvant includes one or more of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0014] The present invention provides the use of Farnesol in preparing a reagent for reducing STING phosphorylation.

[0015] Furthermore, reducing STING phosphorylation refers to reducing the phosphorylation level of STING protein.

[0016] Furthermore, the reduction of STING phosphorylation occurs when the detected cells have been infected with pathogens.

[0017] Furthermore, the cells include epithelial cells.

[0018] Furthermore, the cells include vaginal epithelial cells.

[0019] Furthermore, the pathogen is vulvovaginal Candida.

[0020] The present invention provides use of Farnesol in preparing a STING expression regulator.

[0021] The present invention provides the use of Farnesol and a vector overexpressing the STING protein in preparing a product for inhibiting the adhesion of vulvovaginal Candida spores to cells.

[0022] Furthermore, the vector includes a viral or non-viral vector.

[0023] Furthermore, the vector is capable of overexpressing STING protein.

[0024] Furthermore, the vector can be transduced into cells and expressed.

[0025] Furthermore, the expression refers to the transcription and translation of a nucleotide sequence into a protein / amino acid sequence.

[0026] Furthermore, the cells include epithelial cells.

[0027] Furthermore, the cells include vaginal epithelial cells.

[0028] Furthermore, the products include primers, probes, chips, test kits, nucleic acid membrane strips, test papers, vaccines, and drugs.

[0029] The present invention provides the use of Farnesol and a vector overexpressing STING protein in preparing a product for inhibiting the growth of vulvovaginal Candida on cells.

[0030] Furthermore, the vector includes a viral or non-viral vector.

[0031] Furthermore, the vector is capable of overexpressing STING protein.

[0032] Furthermore, the vector can be transduced into cells and expressed.

[0033] Furthermore, the expression refers to the transcription and translation of a nucleotide sequence into a protein / amino acid sequence.

[0034] Furthermore, the vector for overexpressing the STING protein is intended to transduce a nucleotide sequence capable of overexpressing the STING protein into cells and express it within the cells.

[0035] Furthermore, the cells include epithelial cells.

[0036] Furthermore, the cells include vaginal epithelial cells.

[0037] Furthermore, the products include primers, probes, chips, test kits, nucleic acid membrane strips, test papers, vaccines, and drugs.

[0038] The present invention provides the use of Farnesol and a vector overexpressing a STING protein in preparing a product for increasing the level of IgG antibodies in cells.

[0039] Furthermore, the vector includes a viral or non-viral vector.

[0040] Furthermore, the vector is capable of overexpressing STING protein.

[0041] Furthermore, the vector can be transduced into cells and expressed.

[0042] Furthermore, the expression refers to the transcription and translation of a nucleotide sequence into a protein / amino acid sequence.

[0043] Furthermore, the cells include immune cells.

[0044] Furthermore, the immune cells include T cells, B cells, NK cells, NKT cells, DNT cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor-infiltrating lymphocytes.

[0045] Furthermore, the products include primers, probes, chips, test kits, nucleic acid membrane strips, test papers, vaccines, and drugs.

[0046] In some embodiments, the Farnesol and the vector overexpressing the STING protein exert a synergistic effect in inhibiting the adhesion and growth of vulvovaginal Candida spores.

[0047] The present invention provides a method for screening compounds that affect Farnesol's inhibition of STING phosphorylation, the method comprising:

[0048] A1. Add the test compound to the system containing Farnesol and in vitro cells;

[0049] A2. Detect the phosphorylation level of STING;

[0050] comparing the phosphorylation level obtained in A2 with the phosphorylation level obtained in the absence of the test compound; if the phosphorylation level obtained in A2 is higher than the phosphorylation level obtained in the absence of the test compound, identifying the test compound as a compound that promotes Farnesol's inhibition of STING phosphorylation;

[0051] The phosphorylation level obtained in A2 is compared with the phosphorylation level obtained in the absence of the test compound. If the phosphorylation level obtained in A2 is lower than the phosphorylation level obtained in the absence of the test compound, the test compound is identified as a compound that inhibits Farnesol's inhibition of STING phosphorylation.

[0052] In some embodiments, the effect of Farnesol on the inhibition of STING phosphorylation is divided into two types: promoting Farnesol's inhibition of STING phosphorylation and inhibiting Farnesol's inhibition of STING phosphorylation, and is applied to the regulation of STING protein phosphorylation in cells in vitro.

[0053] In some embodiments, the compound includes but is not limited to oxides, acids, bases, salts, and organic matter. In some embodiments, the oxide is a compound composed of two elements, one of which is oxygen. In some embodiments, the acid is a compound in which all the cations generated upon ionization are hydrogen ions. In some embodiments, the base is a compound in which all the anions generated upon ionization are hydroxide ions. In some embodiments, the salt is a compound that generates metal cations (or ammonium ions) and acid ions upon ionization. In some embodiments, the organic matter is a general term for carbon-containing compounds (excluding carbon monoxide, carbon dioxide, carbonates, metal carbides, and cyanides) or hydrocarbons and their derivatives.

[0054] As used herein, the term "salt" includes pharmaceutically acceptable salts, including salts prepared from pharmaceutically acceptable bases or acids. In some embodiments, the "acid" is a pharmaceutically acceptable acid, including but not limited to inorganic and organic acids, such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinylsulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid and p-toluenesulfonic acid. In some embodiments, the "base" is a pharmaceutically acceptable base, including but not limited to acceptable inorganic bases including sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, magnesium and aluminum. Pharmaceutically acceptable organic non-toxic bases include primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines, cyclic amines and basic ion exchange resins) such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine and polyamine resins.

[0055] Biologically acceptable salts can involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a biologically acceptable salt can have more than one charged atom in its structure. In the case where multiple charged atoms are part of a biologically acceptable salt, there can be multiple counterions. Thus, a biological salt can have one or more charged atoms and / or one or more counterions.

[0056] The present invention provides a method for regulating STING expression in in vitro cells, comprising adding Farnesol to the in vitro cells.

[0057] Furthermore, the cells include epithelial cells.

[0058] Furthermore, the epithelial cells include vaginal epithelial cells.

[0059] The present invention provides a method for regulating STING phosphorylation in cells in vitro, comprising adding Farnesol to the cells in vitro.

[0060] Furthermore, the cells include epithelial cells.

[0061] Furthermore, the epithelial cells include vaginal epithelial cells.

[0062] The present invention provides a method for screening drugs that affect the enhancing effect of Farnesol on STING expression, the screening method comprising:

[0063] B1. Add the test compound to the cells treated with Farnesol;

[0064] B2. Detect the expression level of STING in cells:

[0065] comparing the expression level obtained in B2 with the expression level obtained in the absence of the test compound; if the expression level obtained in B2 is higher than the expression level obtained in the absence of the test compound, identifying the test compound as a drug that promotes the enhancing effect of Farnesol on STING expression;

[0066] The expression level obtained in B2 is compared with the expression level obtained in the absence of the test compound. If the expression level obtained in B2 is lower than the expression level obtained in the absence of the test compound, the test compound is identified as a drug that inhibits the enhancing effect of Farnesol on STING expression.

[0067] Furthermore, the cells include epithelial cells.

[0068] Furthermore, the epithelial cells include vaginal epithelial cells.

[0069] Furthermore, the compounds include small molecule compounds and macromolecular compounds.

[0070] Furthermore, the compound includes protein analogs, antibodies, DNA, and RNA.

[0071] Furthermore, the DNA includes single-stranded DNA, closed-circular DNA, and linked DNA.

[0072] Furthermore, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, RNA with catalytic activity, and various viral RNAs.

[0073] Furthermore, the DNA includes DNA of various conformations, specifically including A-type, B-type, C-type, D-type, E-type, H-type, L-type, P-type, and Z-type.

[0074] Furthermore, the antibody includes dAb, Fab, Fab', scFv, Fv, disulfide-bonded Fv, or contains a single immunoglobulin variable domain.

[0075] In some embodiments, the antibody, such as a VH or VL domain, is specific for NFL protein binding and is monovalent.

[0076] Furthermore, the protein analogs include proteins artificially synthesized through protein engineering.

[0077] Furthermore, the protein analogs include proteins that are artificially synthesized by protein engineering using DNA to predict the mRNA that translates the protein based on the protein function and structure, and then the DNA.

[0078] As used herein, the term "protein analog" refers to a synthetic protein in which one or more amino acids have been genetically and / or chemically modified and which retains the biological activity of the parent protein, such as cytopathic effect or antiproliferative activity. Such biological activity is manifested in a specific biological activity.

[0079] Furthermore, the drug also includes a pharmaceutically acceptable adjuvant.

[0080] Furthermore, the pharmaceutically acceptable adjuvant includes one or more of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0081] In some embodiments, these pharmaceutical excipients are used to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell when oral administration. The preparations that can be used in such drugs can be in the form of the original compound itself or optionally in the form of a pharmaceutically acceptable salt. The drugs thus formulated can be administered in any appropriate manner known to those skilled in the art as needed.

[0082] Furthermore, the dosage form of the drug includes granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions.

[0083] Furthermore, the drug is administered through epidermal administration, inhalation administration, enema administration, ocular administration, nasal administration, digestive tract administration, oral administration, intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, and bone marrow injection.

[0084] In some embodiments, the drug can be manufactured by methods well known in the art, such as conventional granulation, mixing, dissolution, encapsulation, lyophilization or emulsification, etc. The drug can be prepared in various forms, including granules, precipitates or microparticles, powders (including lyophilized powders, rotary dried powders or spray dried powders, amorphous powders), tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions.

[0085] The present invention provides a method for increasing the level of IgG antibodies in cells in vitro for non-therapeutic purposes, comprising the steps of using Farnesol and a vector overexpressing STING protein.

[0086] Furthermore, the cells include immune cells.

[0087] Furthermore, the immune cells include T cells, B cells, NK cells, NKT cells, DNT cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor-infiltrating lymphocytes.

[0088] The present invention provides a computer-assisted drug screening system based on Farnesol and STING, comprising:

[0089] Reaction unit: Add test compounds to cells that have been treated with Farnesol;

[0090] Detection unit: detect the expression level of STING in cells;

[0091] Determination unit: comparing the expression level obtained with the expression level obtained in the absence of the test compound; if the expression level obtained in the detection unit is higher than the expression level obtained in the absence of the test compound, identifying the test compound as a drug that promotes the enhancement effect of Farnesol on STING expression; comparing the expression level obtained with the expression level obtained in the absence of the test compound; if the expression level obtained is lower than the expression level obtained in the absence of the test compound, identifying the test compound as a drug that inhibits the enhancement effect of Farnesol on STING expression;

[0092] Further, the cells include epithelial cells;

[0093] Furthermore, the epithelial cells include vaginal epithelial cells.

[0094] The term "unit" used in the present invention refers to an application-specific integrated circuit (ASIC), an electronic circuit, a processor (such as a shared processor, a proprietary processor or a group processor, etc.) and a memory for executing one or more software or firmware programs, a merging logic circuit and / or other suitable components that support the functions described. However, the term "unit" is not limited to software or hardware. The term "unit" can be an element that can be configured to be included in an addressable storage medium or can be configured to reproduce one or more processors. Therefore, the example of the term "unit" can include elements (such as software elements, object-oriented software elements, class elements, and task elements), processors, functions, attributes, procedures, subroutines, program code segments, drivers, firmware, microcodes, circuits, data, databases, data structures, tables, arrays, and variables. A small amount of elements and units can be used to combine the functions provided in an element or unit, or other elements and units can be used to subdivide the functions provided in an element and unit.

[0095] The present invention provides a computer device, comprising: a memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, any of the above methods is implemented.

[0096] The term "device" is not limited to one or a specific number of physical objects (e.g., a smartphone, a controller, a processing system, etc.). As used herein, a device can be any device having one or more components that can implement at least some portions of the present invention. Although the following description and examples use the term "device" to describe various aspects of the present invention, the term "device" is not limited to a specific configuration, type, or number of objects.

[0097] The term "processor" refers to any type of processor, such as a microprocessor, an embedded processor, a digital signal processor (DSP), a network processor, or other device for executing code, and may include more than one processor, such as a multi-core design or multiple processors each having a multi-core design. The processor can be configured to execute a sequence of computer program instructions, such as a sequence of those instructions stored in a memory, to perform various operations, processes, and methods according to the exemplary embodiments of the present invention.

[0098] The term "memory" refers to any type of long-term, short-term, volatile, non-volatile or other memory and should not be limited to any particular type of memory or any particular number of memories or types of media storing memory. The term "memory" can be any suitable memory element (e.g., random access memory (RAM), read-only memory (ROM), erasable programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), application specific integrated circuit (ASIC), etc.), software, hardware, firmware, or any other suitable component, device, element or object, as appropriate and based on the particular needs.

[0099] The present invention provides a computer-readable storage medium having a computer program thereon, comprising: when the computer program is executed by a processor, implementing any of the above methods.

[0100] Advantages and beneficial effects of the present invention:

[0101] The present invention discovered that Farnesol can regulate the level of STING protein, thereby treating vulvovaginal candidiasis. The present invention provides a method for screening drugs that can assist in regulating Farnesol and STING proteins, which can help find more compounds that can promote or inhibit the functions of Farnesol and STING proteins and has excellent market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 This is a graph showing the efficiency of siRNA interference with the STING gene in VK2 / E6E7 cells;

[0103] Figure 2 This is a graph showing the effect of overexpressing STING;

[0104] Figure 3 This is a colony PCR run gel image;

[0105] Figure 4 This is the overall result of Gram staining in the adhesion inhibition experiment;

[0106] Figure 5 This is a graph showing the effect of Farnesol on spore adhesion;

[0107] Figure 6 This is a diagram showing the effect of STING interference on spore adhesion;

[0108] Figure 7 This is a diagram showing the effect of STING overexpression on spore adhesion;

[0109] Figure 8 This is a diagram showing the effect of STING interference on the inhibition of fungal adhesion by farnesol;

[0110] Figure 9 This figure shows the effect of STING overexpression on the inhibition of fungal adhesion by farnesol;

[0111] Figure 10 This is the result of the crystal violet method for testing fungal activity;

[0112] Figure 11 This is a graph showing the effect of Farnesol on the anti-Candida albicans activity of VEC;

[0113] Figure 12 This is a diagram showing the effect of STING interference on the anti-Candida albicans activity of VEC;

[0114] Figure 13 This figure shows the effect of STING overexpression on the anti-Candida albicans activity of VEC;

[0115] Figure 14 This is a graph showing the effect of STING interference on the anti-candida albicans activity of farnesol-promoted VEC;

[0116] Figure 15 This figure shows the effect of STING overexpression on the anti-candida albicans activity of farnesol-promoted VEC;

[0117] Figure 16 This is a diagram showing the regulatory effect of Farnesol on the secretion of immune active factors by VEC;

[0118] Figure 17 This is a diagram showing the regulatory effect of STING interference on VEC secretion of immune active factors;

[0119] Figure 18 This is a diagram showing the regulatory effect of STING overexpression on VEC secretion of immune active factors;

[0120] Figure 19 This is a diagram showing the effect of STING interference on the immunomodulatory effect of farnesol;

[0121] Figure 20 This is a diagram showing the effect of STING overexpression on the immunomodulatory effect of farnesol;

[0122] Figure 21 This is a diagram showing the effect of Farnesol on the immune regulation after STING interference / overexpression;

[0123] Figure 22 These are the morphological features of normal VK2 / E6E7 cells under optical microscopy (×200) and scanning electron microscopy (×2000);

[0124] Figure 23 is the scanning electron microscopic morphological characteristics of VK2 / E6E7 cells 6 h after infection (×2000, ×10000);

[0125] Figure 24 This is the scanning electron microscopy result of VEC infected with Candida albicans;

[0126] Figure 25 is the scanning electron microscopic morphological characteristics of VK2 / E6E7 cells 6 h after infection (×3000);

[0127] Figure 26 is the scanning electron microscopic morphological characteristics of VK2 / E6E7 cells 12 h after infection (×2000);

[0128] Figure 27 Figure 2 shows the effects of Farnesol on the morphology and ultrastructure of VK2 / E6E7 and Candida albicans (×2000, ×3000).

[0129] Figure 28 This is a statistical result graph of the effect of Farnesol on the interaction between VEC and Candida albicans;

[0130] Figure 29 This is the total result chart of the LIVE / DEAD fungus test kit;

[0131] Figure 30 This is a graph of Western Blot experimental results;

[0132] Figure 31 This is a diagram showing the role of the STING signaling pathway in fungal infection and farnesol antifungal immunity;

[0133] Figure 32 This is a schematic diagram of the rat VVC model establishment and experimental process;

[0134] Figure 33 This is the HE staining result;

[0135] Figure 34 is the immunohistochemistry results of IFN-γ, IL-4, IL-17, and Non B-IgG;

[0136] Figure 35 This is the statistical result graph of the total score of STING and IFN-αIRS;

[0137] Figure 36 This is the result of immunohistochemistry of STING and IFN-α;

[0138] Figure 37 This is the transmission electron microscopy observation result of rat vaginal tissue. DETAILED DESCRIPTION

[0139] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. It should be understood that the embodiments described in the present invention are only a part of the embodiments that can be implemented in the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0140] Example 1 Experimental Materials and Experimental Methods

[0141] 1. Experimental Materials

[0142] Experimental cells: human vaginal epithelial cells (VEC) VK2 / E6E7 cells.

[0143] Experimental strain: Candida albicans ATCC96113.

[0144] Experimental reagents: farnesol: trans, trans-Farnesol (Sigma-Aldrich 277541, CAS No. 106-28-5), Gram stain solution.

[0145] Experimental kit: IFN-α, IFN-β, IFN-γ, IL-4, IL-17, IgA, IgG.

[0146] 2. Experimental methods

[0147] 2.1 Treatment of Candida albicans:

[0148] Inoculate ATCC96113 Candida albicans standard strain into a weak medium and incubate at 30-35°C. Subculture three times. Resuspend the Candida albicans in RPMI 1640 and adjust the concentration to a desired level after counting using a hemocytometer. Store at 4°C until ready for use. It is best to prepare the fungal suspension the same day it is used.

[0149] 2.2 Co-culture of cells with Candida albicans:

[0150] 12-well culture plates were inoculated with 1 mL of 1×10 5 Incubate the cells with a standard VK2 / E6E7 cell suspension (100 cells / mL) for 24 hours. Add 1 mL of Candida albicans suspension to each well and incubate in a 30-35°C, 5% CO2 incubator for 1 hour. Remove the coverslips, rinse three times with PBS, air-dry, and then fix with methanol for 30 minutes. Gram stain and count 30 epithelial cells randomly under an oil immersion microscope (×1000) to calculate the average adhesion index for each epithelial cell.

[0151] 2.3 Construction of STING overexpression vector

[0152] 2.3.1 Gene information: STING1, Gene ID: 340061, a total of 3 transcripts, temporarily using the longest transcript NM_198282.4, a total of 1140bp.

[0153] 2.3.2 Vector Construction: Purchase a cDNA fragment of the STING gene from Youbao. Design primers targeting specific restriction sites. Use the cDNA as a template for PCR amplification of the target fragment, which is then ligated into a specific overexpression vector. Select positive clones for further expansion and plasmid extraction.

[0154] 2.3.3 Plasmid identification: The extracted plasmid was transfected into human vaginal epithelial cells (VEC) VK2 / E6E7 cells. After overexpression for 24-48 hours, samples were collected for detection.

[0155] 2.4STING siRNA targeted screening

[0156] Three STING gene siRNAs and one NC siRNA were constructed and transfected into human vaginal epithelial (VEC) VK2 / E6E7 cells. STING gene mRNA expression was assessed by Q-PCR (experimental groups: normal cells, NC, and three siRNAs; no biological replicates). Based on the experimental results, the siRNA with the best interference efficiency was selected for vector construction and subsequent experiments.

[0157] 2.4.1 Interference sequence information

[0158] Using NM_198282.4 (see https: / / www.ncbi.nlm.nih.gov / gene / 340061) as the target, three siRNA sequences were designed according to the Tuschl rule, as follows:

[0159] STING-homo-831 sequence:

[0160] sense(5'-3'): CCGGAUUCGAACUUACAAUTT,

[0161] antisense(5'-3'):AUUGUAAGUUCGAAUCCGGTT.

[0162] STING-homo-775 sequence:

[0163] sense(5'-3'):CUGGCAUGGUCAUAUUACATT,

[0164] antisense(5'-3'):UGUAAUAUGACCAUGCCAGTT.

[0165] STING-homo-1001 sequence:

[0166] sense(5'-3'):GCAUCAAGGAUCGGGUUUATT,

[0167] antisense(5'-3'): UAAACCCGAUCCUUGAUGCTT.

[0168] 2.4.2 Cell transfection

[0169] 2.4.2.1 Take VK2 / E6E7 cells in the logarithmic growth phase and in good growth condition, make a single cell suspension with KSF-M medium, and plate 2×10 cells per well. 5 (1) The cells were evenly seeded into 6-well plates and cultured overnight at 37°C in a saturated humidity of 5% CO2; (2) 2 hours before transfection, the medium was changed to serum-free Opti-MEM medium; (3) The cells were transfected according to the following experimental groups: 1) VK2 / E6E7 cells; 2) VK2 / E6E7 cells + STING-homo-831; 3) VK2 / E6E7 cells + STING-homo-775; 4) VK2 / E6E7 cells + STING-homo-1001.

[0170] 2.4.2.2 Transfection Steps: For each transfection sample, prepare as follows:

[0171] a. Dilute 10 μL of each small fragment with 100 μL of serum-free opti-MEM, mix gently with a pipette tip, and let stand at room temperature for 5 minutes. b. Gently mix Lipofectamine™ 2000 before use, then dilute 5 μL of Lipofectamine™ 2000 in 100 μL of opti-MEM and let stand at room temperature for 5 minutes. c. After standing at room temperature for 5 minutes, mix Lipofectamine™ 2000 and the small fragment dilution (total volume 200 μL), mix gently, and let stand at room temperature for 20 minutes.

[0172] 2.4.2.3 Add 200 μL of the mixture to each well and gently shake the cell culture plate back and forth to mix the mixture with the culture medium in the culture plate.

[0173] 2.4.2.4Cultivate cells in a 37°C CO2 incubator. After 6 hours, aspirate the transfection solution and replace it with normal culture medium.

[0174] 2.4.2.5 Cell processing: 48 hours after cell transfection, cells were directly collected for PCR detection.

[0175] 2.5 Real-time fluorescence quantitative PCR detection

[0176] 2.5.1 RNA extraction using Trizol method.

[0177] 2.5.2 RT reverse transcription into cDNA: Reverse transcription reaction system: (two-step method).

[0178] Reverse transcription reaction system: RT1: After removing genomic DNA, the reaction was carried out at 42°C for 2 min using a system consisting of 5 μg of template RNA, 4 μL of 4×gDNA wiper mix, and 16 μL of RNase-free ddH2O. RT2: A reverse transcription reaction system was prepared using 4 μL of 5×HiScript II Select qRT SuperMix II and 16 μL of RT1 reaction solution at 50°C for 15 min, 85°C for 5 s, and 4°C for 10 min.

[0179] 2.5.3 Real-time fluorescence quantitative PCR detection.

[0180] 2.6 Fungal adhesion assay: Calculation of the Candida albicans adhesion index

[0181] 12-well culture plates were inoculated with 1 mL of 1×10 5 Standard VK2 / E6E7, STING(+)VK2 / E6E7, and STING(-)VK2 / E6E7 cell suspensions were incubated for 24 hours. 1 mL of Candida albicans suspension was added to each well and incubated at 37°C in a 5% CO2 incubator for 1 hour. After 1 hour of infection, 1 mL of 50 μM farnesol was added and incubated for 1 hour before terminating the culture. The coverslips were removed, rinsed three times with PBS, air-dried, and fixed with methanol for 30 minutes. Gram staining was performed, and 30 epithelial cells were randomly counted under an oil immersion microscope (×1000). The average adhesion index of each epithelial cell was calculated.

[0182] 100 μL of 5×10 3 CFU / mL standard VK2 / E6E7, STING(+)VK2 / E6E7, and STING(-)VK2 / E6E7 cell suspensions were incubated at 37°C, 5% CO2 for 24 hours. The culture supernatant in the wells was aspirated and discarded, and after washing twice with PBS, 100 μL of 2x 50 μM farnesol was added according to the concentration gradient and incubated for 6 hours before discarding the supernatant. The inoculation concentration in each well was 5×10 3Incubate 100 μL of the CFU / mL bacterial suspension for 24 hours. Add 100 μL of 0.1% crystal violet solution to each well and incubate at 37°C for 30 minutes. Rinse slowly with sterile PBS three times, then add 100 μL of 95% ethanol for 5 minutes. Transfer the wells to another 96-well plate for detection. Measure the absorbance of each well at 595 nm using a microplate reader. Repeat each experiment at least three times.

[0183] 2.7LIVE / DEAD fluorescence staining

[0184] 12-well culture plates were inoculated with 1 mL of 5 × 10 5 / mL standard VK2 / E6E7, STING(+)VK2 / E6E7 and STING(-)VK2 / E6E7 cell suspensions were added with equal proportions of Candida albicans culture and cultured at 37℃, 5% CO2 for 24 hours. The cells were then treated with 50μM farnesol and gently washed with PBS three times. The supernatant and non-adherent cells were removed to set up blank control groups. After drying at room temperature, Live / Dead fluorescent dye ( FungaLight TM Yeast Viability Kit, L34952, Invitrogen, USA) fluorescent stain, stain for 15 minutes at room temperature in the dark, and take pictures under a fluorescence microscope. Live / Dead dye includes green fluorescent nucleic acid dye 9 (to mark all fungal cells) and the red fluorescent nucleic acid dye pyridinium iodide (to mark fungal cells with damaged cell membranes). Live fungi show green fluorescence, while dead fungi show red fluorescence. The effects of VEC on the killing of Candida albicans after farnesol treatment and STING interference were observed.

[0185] 2.8 Detect the levels of IFN-α, IFN-β, IFN-γ (Th1 type, type II IFN), IL-4 (Th2 type), IL-17 (Th17 type), and IgA and IgG that can neutralize fungal particles in the cell supernatant.

[0186] 2.9WB experiment

[0187] 2.9.1 Preparation of SDS-PAGE Gel

[0188] (1) Clean the glass plate for filling with gel, dry it and fix it, and determine the mark line for the filling separation gel liquid level (about 0.5-1.0 cm from the bottom of the sample comb). (2) Prepare 12% separation gel and quickly pour it into the gel glass tank until the liquid level reaches the mark line (avoid the formation of bubbles). (3) Immediately cover the gel surface with deionized water (isolate the air to help gel polymerization), and leave it at room temperature for about 40 minutes until the separation gel solidifies. (4) Prepare 5% concentrated gel. (5) Pour out the deionized water covering liquid and absorb the remaining liquid with absorbent paper. Place the gel plate vertically again, gently add 5% concentrated gel solution (be careful to avoid the formation of bubbles), insert the sample comb, and gel at room temperature for about 40 minutes. (6) Gently remove the comb, place the "concave" side of the glass clamp against the electrophoresis tank, and fix the two sides well on the electrophoresis tank with clips.

[0189] 2.9.2 Sample denaturation and electrophoresis

[0190] (1) Take out the extracted total protein sample from the -80℃ freezer and immediately put it in ice (to reduce protein degradation) to wait for it to melt. (2) According to the protein quantification results, add 20 micrograms of the corresponding volume of total protein sample and 5× protein gel electrophoresis loading buffer, mix gently, denature at 95℃ for 10 minutes, and immediately put it in ice for use. (3) Gently add the protein sample to the gel well, set the electrophoresis instrument to the steady-state state, turn on the power supply, adjust the voltage to 80V to allow the sample to pass through the stacking gel and separation gel (voltage is about 8V / cm). Electrophoresis allows the dye to be placed in the appropriate position of the separation gel, and end the electrophoresis.

[0191] 2.9.3 Gel transfer and detection

[0192] After gel electrophoresis, the separated protein bands are transferred to a solid support via transfer electrophoresis. These bands are then incubated and detected with an unlabeled primary antibody and a horseradish peroxidase-labeled secondary antibody. A variety of solid supports are used for Western blotting; this experiment uses a PVDF membrane as the solid support. Wet transfer is employed.

[0193] (1) Pretreatment of PVDF membrane: Soak in 100% methanol for 2-3 minutes, rinse with water and electrotransfer solution for 2 minutes × 2 times, and place in electrotransfer solution for later use; (2) Cut 6 layers of filter paper of the same size as the gel, soak them in transfer buffer and set aside; (3) Remove the electrophoresis plate and lay it flat (with the "concave" glass plate at the bottom), carefully remove the gasket in the clamp and remove the upper glass plate, cut off the excess gel, and rinse the sample gel with electrotransfer solution once; (4) Load the sample gel and membrane into the transfer clamp marked with positive and negative poles: starting from the cathode side, in the order of 3 layers of filter paper → sample gel → PVDF membrane → three layers of filter paper (note: eliminate bubbles), fasten the transfer clamp and place it in the transfer electrophoresis tank containing transfer buffer; (5) Connect the transfer electrophoresis line correctly to ensure that the charge flows from the negative pole to the positive pole. (Transfer conditions: other current 130ma, voltage about 60v, transfer time is 60min) (6) Blocking: Carefully remove the transfer membrane and place it in the blocking solution, and block it for 1h at room temperature and slowly shake it on a shaker; (7) Primary antibody reaction: Dilute the primary antibody with blocking solution; directly place the blocked membrane in the primary antibody working solution and react at 4℃ overnight; (8) Wash the membrane: Place the reaction membrane in a plate and wash it three times with 1×TBST (wash slowly at room temperature) for 10 minutes each time to wash away the unbound primary antibody; (9) Secondary antibody reaction: Place the washed primary antibody reaction membrane in the secondary antibody working solution (1:10000) and slowly shake it at room temperature and away from light for 60 minutes; Secondary antibody (goat anti-rabbit, bioeasy, BE0101). (10) Wash the membrane: Wash the membrane with 1×TBST, the same method as (8), to wash away the free secondary antibody; (11) Expose and wash the film; (12) Analyze the grayscale value using image J software.

[0194] Example 2 siRNA interference and overexpression of STING

[0195] Using NM_198282.4 (see https: / / www.ncbi.nlm.nih.gov / gene / 340061) as the target, three siRNA sequences STING-homo-831, 775, and 1001 were designed according to the Tuschl rule. Transient transfection was performed according to the instructions of the LipofectamineTM2000 kit. The test results are shown in the figure. Figure 1 shown.

[0196] Primers were designed using NM_198282.4 (see https: / / www.ncbi.nlm.nih.gov / gene / 340061) as the target sequence, and pUC57-Homo TMEM 173 was used as a template to amplify the target fragment by PCR. The target DNA fragment was digested and ligated with the vector pcDNA3.1-3×Flag-C. Transformed single clones were screened and identified. The results are shown below. Figure 2 As shown. Select Figure 3 The third strain shown was sent for sequencing comparison, and the alignment with human STING was completely correct, indicating that pcDNA3.1-3×Flag-C-HomoTMEM173 was successfully constructed.

[0197] Example 3 Adhesion inhibition experiment

[0198] The adhesion inhibition experiment was performed in groups as follows:

[0199] 1) V: VK2 / E6E7 cells. 2) VC: VK2 / E6E7 cells + Candida albicans. 3) VF: VK2 / E6E7 cells + farnesol. 4) VCF: VK2 / E6E7 cells + Candida albicans + farnesol. 5) VO: VK2 / E6E7 cells + STING-overexpression plasmid. 6) VOC: VK2 / E6E7 cells + STING-overexpression plasmid + Candida albicans. 7) VS: VK2 / E6E7 cells + STING-interference plasmid. 8) VSC: VK2 / E6E7 cells + STING-interference plasmid + Candida albicans. 9) VOCF: VK2 / E6E7 cells + STING-overexpression plasmid + Candida albicans + farnesol. 10) VSCF: VK2 / E6E7 cells + STING-interference plasmid + Candida albicans + farnesol. V: vaginal epithelial cells, C: Candida albicans, F: farnesol, S: interference; O: overexpression.

[0200] VEC was cultured for 24 hours, treated with bacteria for 1 hour, and treated with 50 μM farnesol for 1 hour. Gram staining was performed, and the number of spores adhering to 30 epithelial cells was randomly counted under an oil immersion microscope. The average adhesion index of each epithelial cell was calculated. The total results of the Gram staining of the adhesion inhibition experiment are shown in the figure. Figure 4 shown.

[0201] By counting the average number of spore adhesions, it was found that Farnesol could significantly inhibit the adhesion of Candida albicans spores to vaginal epithelial cells (LSD: P < 0.001). Figure 5 Although the number of adhered spores increased after STING interference compared to the non-interference condition, the difference was not significant (LSD: P = 0.267). Figure 6 As shown, STING interference has no effect on spore adhesion. Overexpression of STING can significantly inhibit the adhesion of Candida albicans spores to vaginal epithelial cells (LSD: P = 0.001). Figure 7As shown in the results, it is suggested that STING overexpression can promote the anti-spore adhesion of vaginal epithelial cells. STING interference can offset the inhibitory effect of farnesol on fungal adhesion (LSD: P = 0.008). The number of adherent spores after interference was not significantly different from that of the untreated infection group (LSD: P = 0.645). Figure 8 As shown in Figure 2. Overexpression of STING significantly enhanced the inhibitory effect of farnesol on fungal adhesion (LSD: P = 0.027). Figure 9 As shown, it suggests that the two can synergistically promote VEC antifungal adhesion.

[0202] Example 4 Fungal proliferation activity experiment

[0203] Crystal violet assay: VEC culture for 24 hours, bacterial treatment for 24 hours, and 50 μM farnesol treatment for 6 hours. Incubate with 0.1% crystal violet solution at 37°C for 30 minutes, followed by 95% alcohol treatment for 5 minutes. Transfer the solution to another 96-well plate and measure the OD. 595 The test results are as follows. Figure 10 shown.

[0204] Calculation formula: relative fungal growth rate (%) = (OD of treatment group - OD of control group) × 100% / OD of blank group.

[0205] By analyzing the relative fungal growth rates, we found that Farnesol could significantly inhibit the growth of fungi, VC: 171.72±10.10 vs. VCF: 113.79±14.37 (LSD: P<0.001). Figure 11 After STING interference, the anti-candida albicans activity of VEC was significantly reduced, VC: 171.72 ± 10.10 vs. VSC: 199.15 ± 8.31 (LSD: P = 0.01). Figure 12 As shown in Figure 2. Overexpression of STING significantly increased the anti-candida albicans activity of VEC, VC 171.72±10.10 vs. VOC 99.99±7.30 (LSD: P < 0.001). Figure 13 STING interference had no effect on the anti-candida albicans activity of farnesol-promoted VEC, VCF: 113.79 ± 14.37 vs. VSCF: 128.34 ± 19.35 (LSD: P = 0.235). Figure 14 As shown in Figure 2. Overexpression of STING significantly enhanced the anti-candida albicans activity of farnesol-promoted VEC, VCF: 113.79 ± 14.37 vs. VOCF: 66.49 ± 20.92 (LSD: P = 0.04). Figure 15 This suggests that the two may play a synergistic role after overexpression.

[0206] Example 5 ELISA detection of immune factors

[0207] Farnesol and STING's regulatory effects on VEC secretion of immune-active cytokines were examined using ELISAs. Specifically, farnesol treatment of vaginal epithelial cells revealed no significant differences in IFNα, IFNβ, IFNγ, IL-4, IL-17, IgA, or IgG cytokines. Following fungal infection of vaginal epithelial cells, IFNα and IFNγ levels were significantly elevated compared with the control group (P = 0.025, P = 0.016), while IgG levels were significantly decreased (P = 0.005). However, after farnesol treatment, IFNα, IFNβ, IFNγ, IL-17, and IgA levels remained similar (P > 0.05). However, IL-4 (a Th2 humoral immune response factor) levels were significantly decreased compared with the infection group (P = 0.038), while IgG levels were significantly increased (P = 0.002), returning to control levels (all P > 0.05). There were no significant differences in IgA, IL-17 and IFNβ among all groups (P>0.05). Figure 16 Farnesol can reduce the Th2 humoral immune response of vaginal epithelial cells and reduce the susceptibility of vaginal epithelial cells. Farnesol may act as an immune activator or adjuvant, upregulating these "protective" antibodies to fight against Candida albicans infection.

[0208] STING interference regulates the secretion of immune active factors by VEC. After the vaginal epithelial cells interfered with STING expression, there was no significant difference in IFNα, IFNβ, IFNγ, IL-4, IL-17, IgA and IgG cytokines. After the vaginal epithelial cells were infected with fungi, the levels of IFNα and IFNγ cytokines were significantly increased compared with the control group, while IgG was significantly decreased (all P < 0.001). However, after STING interference, there was no significant difference in IFNβ, IFNγ, IL-4, IL-17, IgA, and IgG (P > 0.05). Only IFNα was significantly lower than the infected group (P = 0.001) and returned to the control group level (P = 0.554). There was no significant difference in IL-4, IL-17 and IFNβ among all groups (P > 0.05). The results are as follows Figure 17As shown. Under normal conditions, type I interferon (IFNα) and type II interferon (IFNγ) are at low levels. Fungal infection of vaginal epithelial cells can participate in the host's defense against pathogen infection by releasing cells such as type I interferon (IFNα) and type II interferon (IFNγ), inducing inflammatory responses, and recruiting immune cells, and can play a sentinel role in the activation of acquired immunity. STING interference leads to lower levels of IFNα secreted by vaginal epithelial cells after fungal infection than infected cells. This shows that STING is a key upstream regulator of IFNα (type I interferon).

[0209] STING overexpression regulates the secretion of immune active factors by VEC. After vaginal epithelial cells overexpress STING, there is no significant difference in the levels of all cytokines. After vaginal epithelial cells were infected with fungi, the levels of IFNα and IFNγ cytokines were significantly increased compared with the control group (P=0.003; P<0.001), while IgG was significantly decreased (P<0.001). However, after STING overexpression, there was no significant difference in IFNα, IFNβ, IL-4, IFNγ, IL-17 and IgA (P>0.05). IFNγ was significantly lower than that in the infection group (P=0.006) and failed to return to normal levels, while IgG was significantly higher than that in the infection group (P<0.001) and returned to the level of the control group (P>0.05). There was no significant difference in IgA, IL-17 and IFNβ among all groups (P>0.05). The results are as follows Figure 18 As shown in the results, fungal infection significantly increased the expression of IFNα and IFNγ, while significantly decreased IgG. However, STING overexpression did not change the level of type I IFN in vaginal epithelial cells after the above-mentioned fungal infection, but it downregulated the secretion of IFNγ. This suggests that STING overexpression does not affect the function of type I IFN, but can inhibit Th1 cell immune responses. On the other hand, STING overexpression can increase the expression of protective natural antibody IgG.

[0210] The effect of STING interference on the immunomodulatory effect of farnesol. After STING interference in vaginal epithelial cells (VSCF group), IFNβ, IL-4, IFNγ, IgA, and IgG were not significantly different from those in the VCF group (P>0.05), while IFNα and IL-17 were significantly reduced compared with the VCF group (P=0.003; P=0.014). There were no significant differences in IFNβ, IgA, and IL-4 among all groups (P>0.05). Figure 19 Farnesol treatment had no effect on IFNα. STING interference may downregulate IFNα expression in farnesol-treated vaginal epithelial cells, consistent with expected results. Further experimental investigation is needed to investigate the effect of STING on IL-17.

[0211] The effects of STING overexpression on the immunomodulatory effect of farnesol were as follows: Figure 20 As shown, STING overexpression had no effect on the immunomodulatory effect of farnesol.

[0212] Effect of Farnesol on the immunomodulatory effect after STING interference / overexpression. After adding farnesol, (VSCF group vs. VSC group), IFNγ was significantly reduced (P=0.003) and IgG was significantly increased (P<0.001), while there was no significant difference in other cytokines (P>0.05). Figure 21 As shown in Figure 3, after STING knockdown, Farnesol downregulated IFNγ, restoring it to normal levels, and upregulated the expression of protective natural antibodies (IgG) in cells with STING knockdown. Farnesol had no effect on the immune function of cells with STING overexpression.

[0213] Example 6 Scanning electron microscopy results

[0214] Scanning electron microscopy: (1) Specimen preparation: cell slides (2) Fixation (3) Dehydration (4) Drying (5) Spray plating (6) Observation and recording. VEC culture for 24 hours, bacterial culture for 24 hours, and 50 μM farnesol treatment for 24 hours. For each sample, count the number of live cells, dead cells, hyphae, and spores in 30 consecutive fields (×2000) and calculate the average number per field.

[0215] Microscopic observation of normal human vaginal epithelial cell line VK2 / E6E7 Figure 22 As shown in Figure 2, the morphological characteristics of normal VK2 / E6E7 cells under optical microscope (×200) and scanning electron microscope (×2000). The dynamic observation results of VEC infected with Candida albicans under scanning electron microscope are shown in Figure 2. Figure 23 The morphological characteristics of VK2 / E6E7 cells 6 hours after infection were observed under scanning electron microscopy (×2000, ×10000). Figure 24 As shown in Figure 2, in the early stage of infection, epithelial cells are induced to form pseudopodia / microvilli, but in the late stage of infection, there is no need to induce epithelial cells to form pseudopodia. Figure 25 The scanning electron microscopic morphological characteristics of VK2 / E6E7 cells 6 hours after infection (×3000) are shown in Figure 2. The dynamic observation results of VEC infected with Candida albicans by scanning electron microscopy are shown in Figure 3. Figure 26 The morphological characteristics of VK2 / E6E7 cells 12 hours after infection were shown in the scanning electron microscope (×2000). Figure 27As shown, the effects of Farnesol on the morphology and ultrastructure of VK2 / E6E7 and Candida albicans (×2000, ×3000).

[0216] The mean number of live cells, dead cells, hyphae and spores of VK2 / E6E7 (SEM, ×2000) was counted and the statistical results were as follows: Figure 28 As shown, Candida albicans invades VEC mainly through two mechanisms: inducing endophagy (more common in the early stage) and active penetration (more common in the late stage). VEC, as a non-classical immune cell, has a phagocytic effect on pathogenic fungi and is the body's first line of defense against the invasion of external pathogens. Farnesol reduces the number of fungal spores and hyphae. Farnesol may also have a repair effect on epithelial cells damaged after infection, mainly by maintaining the morphology and activity of VEC and enhancing the phagocytic effect of cells on pathogens after infection.

[0217] Example 7 LIVE / DEAD fungus experiment

[0218] The total results of the LIVE / DEAD fungus test kit are as follows: Figure 29 As shown, dead cells (red fluorescence) and live cells (green fluorescence). VC vs. VCF: After 24 hours of farnesol treatment, the number of dead fungi was significantly increased compared with the model group. VSC / VOC vs. VC: siRNA interference and overexpression of STING had no significant effect on fungal activity. VCF vs. VSCF: After STING knockdown in VEC, the number of dead fungi was significantly reduced after fungal infection and farnesol co-treatment compared with the non-knockdown group. VCF vs. VOCF: After STING overexpression in VEC, the number of dead fungi was significantly increased after fungal infection and farnesol co-treatment compared with the untreated group. Farnesol may exert an antifungal immune response through STING.

[0219] Example 8 Study on the mechanism of STING-I type interferon signaling pathway

[0220] pass Figure 30 、 31Western blot analysis revealed that 24 hours after fungal infection, the phosphorylation levels of STING and TBK1 were significantly increased in Group 2 compared to Group 1 (P < 0.001 and P = 0.027, respectively). Although the total protein expression of TBK1 did not change significantly (P > 0.05), the p-TBK1 / TBK1 level was significantly higher after fungal infection than in the control group (P = 0.046). After STING knockdown, STING expression was significantly decreased, while p-STING expression was significantly increased. Subsequent fungal stimulation failed to increase the phosphorylation levels of STING and TBK1 in VK2 / E6E7, suggesting that the STING-TBK1-IRF3 signaling pathway may play a key role in the development of VVC. Furthermore, combined with ELISA results, STING knockdown significantly reduced IFNα (a type I interferon) in VK2 / E6E7 infected with fungi. Therefore, activation of the STING-TBK1-IRF3-type I interferon signaling pathway may promote the type I interferon response in the early stages of fungal infection. 24 hours after fungal infection, farnesol treatment reversed the low expression of STING protein and the high expression of p-STING protein caused by fungal infection to varying degrees, but had no significant effect on TBK1, p-TBK1, and p-TBK1 / TBK1 (P>0.05). After knocking down STING, farnesol had no significant effect on STING, p-STING, and TBK1 24 hours after fungal infection. Although farnesol treatment for 4 hours significantly reduced the level of p-TBK1 in the infected group, there was no significant difference in p-TBK1 / TBK1 (P>0.05). These results indicate that farnesol can promote the expression of total STING protein and participate in antifungal immune responses through STING-related signaling pathways. However, whether STING acts through downstream TBK1 and IRF3 cascade factors still needs further verification.

[0221] Example 9 Animal Experiment

[0222] A VVC rat infection model was established and treated with farnesol. Nystatin was used as a positive control group. After drug withdrawal, vaginal secretions were examined to calculate the pathogen negative conversion rate. Vaginal tissue was stained with HE and immunohistochemistry, and transmission electron microscopy was used to examine the changes in the ultrastructure of vaginal tissue. The entire animal experimental process was as follows: Figure 32 As shown, the experiment was divided into normal group, model group, Farnesol group, and Nystatin group.

[0223] The experimental results are shown in Table 1. The pathogen conversion rate on the fourth day after drug withdrawal in the farnesol group was higher than that on the first day. There were significant differences in the pathogen conversion rate on the fourth day after drug withdrawal in the infection model group, the Farnesol group, and the nystatin group (Fisher's exact probability method χ 2=17.154, P < 0.0001). There was no significant difference in the negative conversion rate between the farnesol group and the nystatin group, suggesting that farnesol can effectively eliminate Candida albicans pathogens and its antifungal activity is similar to that of the typical antifungal drug nystatin.

[0224] Table 1

[0225]

[0226] The pathological characteristics of different groups were scored, and the results are shown in Table 2. Higher scores indicate a higher degree of pathological abnormality. Compared with the model group (Model): *P<0.05; **P<0.01; ***P 0.001. Farnesol has a similar effect on inflammation repair as nystatin in the treatment of VVC. The relevant HE staining results are shown in Figure 33 shown.

[0227] Table 2

[0228] Grouping inflammation edema Necrosis foaming Microabscess Bleeding Total score Normal group 0.33** 0.17 0.00 0.00 0.00 0.00 0.50** Model Group 2.00 0.50 0.50 0.67 0.00 0.17 3.83 Farnesol Group 0.5** 0.17 0.17 0.33 0.00 0.00 1.17** Nystatin group 0.83** 0.00 0.00 0.17 0.00 0.17 1.17** F-number 4.959 1.667 2.857 2.778 - 0.667 4.521 P-value 0.010 0.206 0.063 0.068 - 0.582 0.014

[0229] Immunohistochemistry was performed on different groups, and the results were as follows Figure 34 The positive percentage score SP, staining intensity score SI, IRS total score, F value, and P value were also calculated. The results are shown in Table 3.

[0230] Table 3

[0231]

[0232]

[0233] These results show that after adding farnesol treatment, no significant changes were observed in IFN-γ and IL-17 levels. Although there were no significant differences compared to the model and nystatin groups, this at least suggests that farnesol does not affect the antifungal activity of Th1 and Th17 cellular immune responses. IL-4, a representative Th2 cytokine, reflects changes in the humoral immune response. There were no significant differences. IgG expression is significantly decreased during fungal infection, and farnesol may help restore non-B IgG expression, thereby supporting local epithelial immunity.

[0234] The total IRS scores, F values, and P values ​​of STING and IFN-α in different groups were statistically analyzed, and the results are shown in Table 4 and Figures 35-36As shown, Candida albicans weakens the host's innate immune response during genital tract infection. STING deficiency reduces the body's defenses against fungal infections, leading to extensive fungal proliferation and invasion in the vagina, exacerbating the infection. Farnesol can promote STING expression, and animal studies have shown that this can reduce inflammation at the cellular level in vitro.

[0235] Table 4

[0236]

[0237] Ultrastructural changes were observed by transmission electron microscopy. Figure 37 Figures (A) show the normal group (6000×), B the model group (6000×), C the farnesol group (3000×), and D the farnesol group (15000×). In the model group, vaginal epithelial mitochondria exhibited blurred structure, swelling, vacuolar changes, and ruptured cristae. Black triangular arrows indicate mitochondria, yellow arrows indicate spores, and long black arrows indicate desmosomes. Farnesol has a repairing effect on vaginal mucosal tissue damaged by infection, primarily by restoring mitochondrial morphology and activity and enhancing intercellular connectivity, thereby enhancing local immune function in the vaginal mucosa from the host's perspective.

Claims

1. A pharmaceutical composition comprising Farnesol and a vector that overexpresses STING protein; The vector for overexpressing the STING protein comprises a nucleotide sequence for expressing the STING protein, and the accession number of the nucleotide sequence for expressing the STING protein in the NCBI GenBank database is NM_198282.

4.

2. The pharmaceutical composition according to claim 1, characterized in that The vector includes viral or non-viral vectors.

3. Use of Farnesol and a vector overexpressing the STING protein in the preparation of a product that inhibits the adhesion of Candida vulvovaginalis spores to cells; The vector for overexpressing the STING protein comprises a nucleotide sequence for expressing the STING protein, and the accession number of the nucleotide sequence for expressing the STING protein in the NCBI GenBank database is NM_198282.

4.

4. Use of Farnesol and a vector overexpressing the STING protein in the preparation of a product for inhibiting the growth of Candida vulvovaginalis on cells; The vector for overexpressing the STING protein comprises a nucleotide sequence for expressing the STING protein, and the accession number of the nucleotide sequence for expressing the STING protein in the NCBI GenBank database is NM_198282.4.

Images