Use of dihydroeriodictyol c

Abstract

The application discloses a use of dihydrochrysosplenetin C, and the use refers to using one or more of dihydrochrysosplenetin C or pharmaceutically acceptable salts thereof as an active ingredient to prepare an anti-inflammatory drug, and the drug has an immunoregulation effect. Experimental results show that the dihydrochrysosplenetin C can reduce excessive immune responses of a body to infection or induction, reduce difficult-to-control critical symptoms in sepsis treatment, and has medicinal value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the anti-inflammatory and immunomodulatory uses of dihydroquinone C. Background Technology

[0002] Excessive inflammatory response is increasingly recognized as a key factor in sepsis-related deaths, especially after discharge. According to the third edition of the international consensus definition of sepsis and septic shock, sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. It is a clinical disease with complex immunopathophysiology, during which inflammation and immunosuppression may occur sequentially or simultaneously. In the early stages of systemic inflammatory response, if the immune system promptly clears pathogens, immune balance can be rapidly restored. Failure to clear pathogens in a timely manner leads to immune dysregulation. In this situation, patients are prone to secondary infections, resulting in chronic immunosuppression, immune failure, and even physical disability, also known as persistent inflammatory-immunosuppressive-catabolic syndrome. This is also common in critical illnesses caused by other non-infectious injuries, such as secondary major trauma and pancreatitis.

[0003] Because excessive inflammatory responses and immunosuppression do not occur independently, they often coexist in the pathological process of sepsis. Therefore, sepsis is difficult to treat, is the most common cause of death in hospitalizations and intensive care units (ICUs), and is also one of the leading causes of long-term mortality in patients with severe trauma. The mortality rate for sepsis survivors is 15% in the first year after discharge and 6.8% in the following five years. In recent years, treatment methods combining anti-inflammatory and immunomodulatory therapies have become a research hotspot. For example, immunomodulatory therapy using ulinastatin (UTI) and Tα1 can improve organ function and reduce mortality in patients with severe sepsis. However, the combination of drugs, optimal dosage, and duration of treatment still require further confirmation through clinical studies. Furthermore, the excessive immune response generated by the body during severe infection is itself a significant factor contributing to the high mortality rate of sepsis; therefore, controlling the excessive immune response is also an effective direction for treatment.

[0004] Dihydro-Zaluzanin C (3-epi-Zaluzanin C, isozaluzanin C, DHZD) is a sesquiterpene compound (Li, C.; Yu, X.; Lei, X. "A Biomimetic Total Synthesis of (+)-Ainsliadimer A" Org. Lett. 2010, 12, 4284-4287.). Dihydro-Zaluzanin C (as shown in Compound 1 below) contains an allyl alcohol structural group and lacks a conjugated system between the corresponding hydroxyl group and the double bond. In contrast, dehydro-Zaluzanin C (as shown in Compound 2 below) contains an α,β-keto structural group, which belongs to a conjugated system. Currently, there are no reports on dihydro-Zaluzanin C as an active ingredient in anti-inflammatory and immunomodulatory drugs.

[0005]

[0006] Although dehydromethionine C has certain anti-inflammatory and antibacterial effects (Chinese patents CN105640937A and CN112587517A), it has not been extensively studied in animal experiments, such as in severe sepsis models, due to its cytotoxicity. Summary of the Invention

[0007] This invention provides a use of dihydroquinone C, which aims to develop the medicinal value of dihydroquinone C in alleviating critical symptoms that are difficult to control in the clinical treatment of diseases such as sepsis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In animal models used to simulate sepsis, experiments showed that dihydromethionine C (DHZD) could significantly protect the body temperature and organs of mice in a septic shock and sepsis model induced by carbapenem-resistant Klebsiella pneumoniae (CRKP). In a mouse septic shock model induced by lethal doses of Gram-negative bacterial lipopolysaccharide (LPS), DHZD could improve the survival rate of mice. Intervention with DHZD could downregulate the expression levels of inflammatory factors and chemokines (such as IL-6, TNF-α, IL-10, and MCP-1) in the serum of septic mice.

[0010] Experiments have shown that DHZD can inhibit the activation of the PI3K / Akt / p70S6K signaling pathway in important innate immune cells (monocytes / macrophages, dendritic cells) involved in the inflammatory response, thereby downregulating the expression levels of cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), chemokines (such as MCP-1), interferon-β (IFN-β), and interleukin-10 (IL-10).

[0011] The above experimental results indicate that DHZD can reduce bodily damage and even death caused by excessive inflammatory responses by inhibiting LPS-induced excessive inflammatory responses. In the treatment of sepsis caused by CRKP infection, DHZD can reduce the excessive immune response to pathogen infection by regulating the host immune response, thus alleviating the difficult-to-control critical symptoms of sepsis. Therefore, the use of dihydroquinone C described in this invention specifically refers to the use of one or more of dihydroquinone C and its stereoisomers, precursor compounds, and pharmaceutically acceptable salts as immunomodulatory active ingredients in the preparation of anti-inflammatory drugs, especially for the preparation of drugs that combine immunomodulation to achieve anti-inflammatory effects.

[0012] As a preferred embodiment, the immunomodulatory active ingredient (e.g., dihydroquinone C) is used alone, or in combination with one or more antibiotics or hormones, wherein the antibiotic is selected from one or more carbapenem antibiotics or quinolone antibiotics, such as meropenem or levofloxacin, and the hormone is selected from the clinically commonly used glucocorticoid dexamethasone.

[0013] As a preferred embodiment, the drug contains, in addition to the active ingredient (e.g., an active ingredient with immunomodulatory effects), a small amount of minor ingredients that do not affect the active ingredient and / or pharmaceutically acceptable carriers. For example, the drug may also contain sweeteners to improve taste, antioxidants to prevent oxidation, and various excipients necessary for formulation.

[0014] As a preferred embodiment, the dosage form of the drug is not limited, as long as it is a dosage form that enables the active ingredient to reach the body. For example, the dosage form of the drug is a common dosage form such as tablets, capsules, powders, granules, pills, syrups, solutions, suspensions, injections, tinctures, oral liquids, aerosols, lozenges, granules, pills, powders, etc., or a sustained-release dosage form such as nano-preparations.

[0015] In the above technical solutions, the "pharmaceuticalally acceptable salt" refers to a salt formed by dihydroquinone C and a pharmaceutically acceptable inorganic or organic acid. The inorganic acid is hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, or sulfuric acid; the organic acid is formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, linaloic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, or amino acids. The term "pharmaceuticalally acceptable" means suitable for humans without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit / risk ratio.

[0016] In the above technical solutions, the "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in a molecule, such as cis-trans isomers, enantiomers, conformational isomers, etc.

[0017] In the above technical solutions, the "precursor compound" refers to a compound that is inactive in vitro but can be metabolized or chemically reacted in vivo to be converted into dihydroquinone C, thereby exerting its pharmacological effects.

[0018] The beneficial effects of this invention are reflected in:

[0019] This invention experimentally reveals that dihydroquinone C can reduce the excessive inflammatory response induced by pathogenic bacterial infection and endotoxins, exert a protective effect on body temperature and organs in sepsis, and improve the survival rate of severe sepsis. Experimental results indicate that dihydroquinone C can be used as an effective component in anti-inflammatory and immunomodulatory drugs (including monotherapy or combined use with antibiotics and hormones to enhance efficacy), possessing significant medicinal value and providing important reference for finding novel candidate drugs and exploring more effective treatment strategies. Attached Figure Description

[0020] Figure 1 Cell viability (A) was measured at 450 nm after co-incubation with different concentrations of DHZD and Raw264.7 cells (12, 24, 48, and 72 h). The viability of Raw264.7 cells (2 × 10⁻⁶ cells) was also measured. 5The secretion of IL-6 (B), TNF-α (C), MCP-1 (D), IFN-β (E), IL-1β (F), and IL-10 (G) in cell culture supernatant was detected by ELISA at different time points (6 and 18 h) after stimulation with different concentrations of DHZD (0, 3, 5, 10, and 15 μM) and LPS (100 ng / mL). (medium was used as a negative control for cultured cells only. Each stimulation experiment was repeated three times, i.e., n=3; *, p<0.05; **, p<0.01; ***, p<0.001).

[0021] Figure 2 Primary mouse peritoneal macrophages (3.5 × 10⁻⁶) 5 Cells were seeded in 24-well plates at 300 μL per cell. On the second day, after stimulation with different concentrations of DHZD (0, 3, 5, 10, and 15 μM) and LPS (100 ng / mL), the cell culture supernatant was collected at different time points (6 and 18 h) to detect the secretion of IL-6 (A), TNF-α (B), MCP-1 (C), IL-1β (D), IFN-β (E), IL-10 (F) and NO (G). (medium was used as a negative control for cultured cells only. Each experiment was repeated three times, i.e., n=3; *, p<0.05; **, p<0.01; ***, p<0.001).

[0022] Figure 3 For BMDCs (with 3.5 × 10 5 BMDCs were seeded in 24-well plates at a density of 300 μL and stimulated with different concentrations of DHZD (0, 3, 5, 10, and 15 μM) and LPS (100 ng / mL) for 6 and 18 h. The secretion of IL-6(A), TNF-α(B), and IL-12p70(C) in the collected cell culture supernatant was then detected. Additionally, co-stimulatory molecules CD40(D), CD86(E), CD80(F), and MHC on the surface of BMDCs were detected by flow cytometry after seeding BMDCs in 12-well plates and stimulation with DHZD (15 μM) and LPS (100 ng / mL) for 24 h, or stimulation with LPS (100 ng / mL) alone for 24 h. Expression of II(Iab)(G) (flow cytometry results were analyzed using FlowJo software; medium was used as a negative control for cultured cells only; each experiment was repeated 3 times, i.e., n=3; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0023] Figure 4The survival rate of female C57BL / 6 mice (randomly divided into 6 groups of 10 mice each) after intraperitoneal injection of 12.5 mg / kg LPS was compared with that of DHZD alone or DHZD combined with DXM (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0024] Figure 5 Female C57BL / 6 mice (nine mice per group) were randomly assigned to five groups (PBS, LPS (10 mg / kg), LPS+DXM (5 mg / kg), LPS+DHZD (40 mg / kg), and LPS+DXM (5 mg / kg)+DHZD (10 mg / kg) groups. The secretion of IL-6 (A), TNF-α (B), MCP-1 (C), and IL-10 (D) in serum (prepared from blood from mouse eyeballs) was detected by ELISA 12 h after drug injection. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0025] Figure 6 For PBS group, CRKP (9×10) 7 Female C57BL / 6 mice (randomly assigned to 7 groups, 10 mice per group) were injected intraperitoneally with a lethal dose of CRKP. The mice were observed every 2-3 hours until 150 hours after the injection of lethal dose of CRKP into the following groups: CFU / mouse group, CRKP+MEM (5 mg / kg) group, CRKP+LVX (75 mg / kg) group, CRKP+DHZD-L (20 mg / kg) group, CRKP+DHZD-H (40 mg / kg) group, and CRKP+MEM (5 mg / kg)+DHZD-L (20 mg / kg) group. The mice were also monitored and their body temperature was monitored and changed before and after modeling (B and C). (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0026] Figure 7 For PBS group, CRKP (6×10) 7 Female C57BL / 6 mice (randomly assigned to 4 groups, 10 mice in each group) were intraperitoneally injected with 6 × 10 CFU / mouse, CRKP+LVX (75 mg / kg), and CRKP+DHZD-H (40 mg / kg). 7 CFUCRKP was performed, and the lungs and livers of mice were fixed and stained with HE after 12 hours (A). The results of pulmonary edema, alveolar infiltration and pulmonary vasculitis were scored according to a 12-point pathological scoring scale (B) (***, p<0.001; ****, p<0.0001).

[0027] Figure 8 Raw264.7 cells (at 1×10⁻⁶) 5 Cells were seeded in 96-well plates at a density of 100 μL and stimulated with DHZD (15 μM) and LPS (100 ng / mL) for 24 h. The cells were then incubated with pHrodo-labeled E. coli in the dark. The results were analyzed by flow cytometry (A) and analyzed using FlowJo (B, C). Each experiment was repeated three times (n = 3). **, p < 0.01; ****, p < 0.0001. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0029] (I) Pharmacological Experiments

[0030] (1) In vitro anti-inflammatory activity of DHZD on Raw264.7 cells

[0031] 1. Sample preparation

[0032] Dihydroquinone C (DHZD) was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg / mL, and further diluted with PBS (phosphate buffered saline) to a stock solution of 40 mg / mL. The solution was then further diluted with serum-free DMEM cell culture medium to prepare sample solutions at concentrations of 3, 5, 10, and 15 μM (a 0 μM sample solution without DHZD was also prepared). Lipopolysaccharide (LPS) at a concentration of 100 ng / mL was used as the stimulus for cell-level experiments.

[0033] 2. Experimental Methods

[0034] 1) Culture of mouse macrophage line Raw264.7 cells

[0035] Subculture the cells in DMEM high-glucose medium containing 10% FBS at 37°C in a 5% CO2 incubator. When the confluence rate reaches 70%–80% as observed under a light microscope, discard the old medium, add 3 mL / 10 mm dish of fresh complete medium, scrape off adherent cells, gently disperse the cell clumps, and then passage the cells according to the specified ratio, or count the cells and plate them for the next experiment.

[0036] 2) Cell proliferation / toxicity detection

[0037] After co-incubating DHZD and Raw264.7 cells at different concentrations, 10 μL of CCK8 enhanced solution was added to each well according to the CCK-8 assay kit instructions. After adding the reagents, the culture plate was gently shaken to aid mixing. The cells were incubated for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula:

[0038] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0039] In the formula, As is the absorbance of the drug test well; Ac is the absorbance of the negative control well; and Ab is the absorbance of the blank well.

[0040] 3) Enzyme-linked immunosorbent assay (ELISA)

[0041] Following the kit instructions, dissolve the standards, capture antibody, and detection antibody, label the dilution factor, aliquot, and store at -20°C. ① Dilute the capture antibody to the working concentration with sterile PBS according to the number of samples and the total number of standards. Add 100 μL / well to the plate using a continuous pipette, coat the ELISA plate, and incubate overnight at room temperature. ② The next day, discard the capture antibody. Add 300 μL of wash buffer (PBS containing 0.05% Tween 20, pH 7.2–7.4) to each well using a multi-channel pipette. After 1 min, blot the wash buffer onto the plate paper. Wash three times, ensuring no wash buffer residue remains in the wells after the final wash. ③ Add reagent diluent (PBS containing 1% BSA, pH 7.2–7.4). 7.2~7.4) Add 300 μL / well, cover with sealing film, and incubate at room temperature for 1 h; ④ Discard the reagent diluent and repeat step 2 for washing; ⑤ Add 100 μL / well of the test sample (cell culture supernatant, appropriately diluted) and standard, cover with sealing film, and incubate at room temperature for 2 h; ⑥ Discard the test sample and standard, and repeat step 2 for washing; ⑦ Dilute the detection antibody to the working concentration with reagent diluent, add 100 μL / well, and incubate at room temperature for 2 h; ⑧ Discard the detection antibody and repeat step 2 for washing; ⑨ Add 100 μL / well of horseradish peroxidase (HRP) conjugated with diluent diluted to the working concentration, and incubate at room temperature in the dark for 20 min; ⑩ Discard the HRP and repeat step 2 for washing; Add 100 μL of the 1:1 prepared substrate solution to each well and react at room temperature in the dark for 20 min. Add 50 μL of stop solution per well, and measure the OD value at 450 nm (detection wavelength) / 570 nm (correction wavelength) using a microplate reader. Calculate the corresponding protein concentration based on the standard curve.

[0042] 3. Measurement and Statistical Methods

[0043] The cytotoxicity of the drug was detected using CCK-8 assay, and the secretion of cytokines in the supernatant was detected using ELISA. Statistical analysis of the results from each group was performed using a t-test, and data were expressed as mean values. ± standard deviation (SD) is expressed.

[0044] 4. Experimental Results

[0045] Experimental results are as follows Figure 1 As shown, in Raw264.7 cells, 0–15 μM DHZD showed no cytotoxicity and did not affect normal cell growth within 72 h. Moreover, DHZD intervention inhibited the secretion of IL-6, TNF-α, MCP-1, IFN-β, IL-1β, and IL-10 stimulated by LPS, exhibiting a significant dose-dependent relationship.

[0046] The results in this section indicate that dihydromethionine C can significantly inhibit the levels of inflammatory factors and chemokines induced by LPS in Raw264.7, and has an anti-inflammatory regulatory effect.

[0047] (2) DHZD inhibits LPS-induced cytokine secretion in primary mouse peritoneal macrophages.

[0048] 1. Sample preparation

[0049] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg / mL, and further diluted with PBS (phosphate buffered saline) to a stock solution of 40 mg / mL. The solution was then further diluted with serum-free DMEM cell culture medium to prepare sample solutions at concentrations of 3, 5, 10, and 15 μM (a 0 μM sample solution without DHZD was also prepared). Lipopolysaccharide (LPS) at a concentration of 100 ng / mL was used as the stimulus for cell-level experiments.

[0050] 2. Experimental Methods

[0051] 1) Induction and culture of primary mouse peritoneal macrophages (pMφ)

[0052] SPF-grade C57BL / 6J female mice (6-8 weeks old) were intraperitoneally injected with 1 mL of sterile 3.5% sodium thioglycolate solution in each abdomen. After 3.5 days, the mice were euthanized by cervical dislocation and disinfected by immersion in 75% ethanol for 5 minutes. The abdominal skin was cut open, and the peritoneal cavity was flushed twice with a 20 mL syringe filled with pre-warmed sterile DMEM. The flushing fluid was collected in a sterile 50 mL centrifuge tube, repeatedly aspirated and pipetted, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in DMEM medium containing 1% antibiotics and 10% FBS. Cell clumps were dispersed using a 10 mL pipette, diluted, counted, and plated. After 2 hours of routine culture, the medium was changed, and the adherent cells were identified as pMφ.

[0053] LPS (100 ng / mL) and different concentrations of DHZD (0–15 μM) were added simultaneously to pMφ cell culture medium, and the cell culture supernatant was collected after treatment at different time points.

[0054] 2) Griess method for determining nitric oxide content

[0055] Follow the instructions in the kit: ① Remove Griess Reagent I, II, and standards, thaw them, and allow them to return to room temperature; ② Dilute the standards with cell culture medium (1–100 μM); ③ Add each standard and sample (cell culture supernatant) to a 96-well plate at 50 μL / well; ④ Add Griess Reagent I reagent to each well at 50 μL / well; ⑤ Add Griess Reagent II reagent to each well at 50 μL / well; ⑥ Gently shake the culture plate to mix, and then measure the absorbance at 540 nm; ⑦ Calculate the concentration of nitric oxide (NO) in the sample based on the standard curve.

[0056] 3. Measurement and Statistical Methods

[0057] ELISA and Griess assays were performed to detect the secretion of inflammatory factors, chemokines, and NO in the cell culture supernatant of each well. The results of each group were statistically analyzed using a t-test, and the data were expressed as mean values. ± standard deviation (SD) is expressed.

[0058] 4. Experimental Results

[0059] Experimental results are as follows Figure 2 As shown, DHZD exhibits anti-inflammatory activity in primary mouse peritoneal macrophages, inhibiting the secretion of LPS-induced pro-inflammatory cytokines IL-6, TNF-α, IL-1β, type I interferon IFN-β, and NO, and reducing the secretion of chemokine MCP-1 and anti-inflammatory cytokine IL-10.

[0060] These results indicate that DHZD downregulates LPS-induced inflammatory responses in macrophages by reducing the production of pro-inflammatory cytokines and chemokines, suggesting that DHZD exhibits good anti-inflammatory activity at the cellular level.

[0061] (3) DHZD inhibits the secretion of inflammatory factors in LPS-induced dendritic cells and reduces the expression of cell surface co-stimulatory molecules.

[0062] 1. Sample preparation

[0063] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg / mL, and further diluted with PBS (phosphate buffered saline) to a stock solution of 40 mg / mL. The solution was then further diluted with serum-free DMEM cell culture medium to prepare sample solutions at concentrations of 3, 5, 10, and 15 μM (a 0 μM sample solution without DHZD was also prepared). Lipopolysaccharide (LPS) at a concentration of 100 ng / mL was used as the stimulus for cell-level experiments.

[0064] 2. Experimental Methods

[0065] 1) Induction and culture of mouse bone marrow-derived dendritic cells (BMDC)

[0066] Four bones (tibia and femur) from the hind limbs of SPF-grade male C57BL / 6J mice (4 weeks old) were collected. Using a 1 mL syringe, pre-warmed 1640 was drawn and repeatedly rinsed from both ends of the bones to extract bone marrow strips until the bone hairs turned white. The bone marrow strips were then dispersed in culture medium using a syringe and centrifuged (1000 rpm × 5 min, RT). The supernatant was discarded, the cell clumps were gently separated, and 2 mL / mouse of erythropoiesis solution was added. Erythropoiesis was stopped at room temperature for 2.5 min, and an equal volume of serum-containing 1640 was added. The cells were centrifuged again (1000 rpm × 5 min, RT), the supernatant was discarded, and the cell clumps were gently separated. 21 mL / mouse of complete DC culture medium (10% FBS 1640 + 50 ng / mL mGM-CSF + 4 ng / mL mIL-4) was added to resuspend the cells. After thorough mixing, 2.5 mL / well was added to each well of a six-well plate and incubated at 37°C with 5% CO2. After 3 days, complete DC culture medium was added, and after 5 days, the cells were ready for counting and plating.

[0067] 2) Flow cytometry (FACS) detection of co-stimulatory molecule expression

[0068] ① Induced mouse bone marrow-derived dendritic cells were prepared into a single-cell suspension and seeded into 12-well plates (5×10⁶ cells / wells). 5 Each well contains 800 μL of LPS (100 ng / mL) and DHZD (15 μM) of the drug.

[0069] ② After co-incubation for 24 hours, gently blow off the cells from each well of the 12-well plate and transfer them to sterile 1.5 mL Epp tubes;

[0070] ③ Low-temperature centrifugation, 1400 rpm, 5 min;

[0071] ④ Discard the supernatant, resuspend in 250 μL PBS, centrifuge and discard the supernatant;

[0072] ⑤ Prepare the live and dead cell staining solution (PBS, Zombie, FcR) according to the instructions, 50 μL / tube, and gently mix the cells (RT, protected from light) for 10 min;

[0073] ⑥ Wash once: Add 220 μL of Fc Buffer per tube and centrifuge at 4℃ (1400 rpm × 5 min);

[0074] ⑦ Discard the supernatant, invert the paper towel, add 50 μL of antibody working solution, shake thoroughly to mix, and incubate at 4°C in the dark for 25 min;

[0075] ⑧ Wash once: Add 200 μL of Fc Buffer per tube, centrifuge at 4°C (1400 rpm × 5 min); after centrifugation, discard the supernatant, invert the paper towel, add 200 μL of PBS, vortex thoroughly to mix, complete cell collection, and prepare for cell loading.

[0076] 3. Measurement and Statistical Methods

[0077] The secretion of inflammatory factors in the cell culture supernatant of each well was detected by ELISA, and the expression of co-stimulatory molecules and MHC class II molecules on the surface of BMDCs was detected by flow cytometry. The results of each group were statistically analyzed using the t-test, and the data were expressed as mean values. ± standard deviation (SD) is expressed.

[0078] 4. Experimental Results

[0079] Experimental results are as follows Figure 3 As shown, DHZD can reduce the secretion of LPS-induced pro-inflammatory cytokines IL-6, TNF-α and IL-12p70, suggesting that DHZD can inhibit the secretion of large amounts of inflammatory factors by LPS-induced dendritic cells; DHZD can significantly inhibit the expression of co-stimulatory molecules and MHC class II molecules, suggesting that DHZD can reduce the ability of dendritic cells to activate T cells.

[0080] (4) DHZD alone or in combination with dexamethasone can improve the survival rate of LPS-induced septic mice.

[0081] 1. Sample preparation

[0082] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 80 mg / mL, and further diluted with sterile PBS to a concentration of 4 mg / mL. Dexamethasone (DXM) solution was prepared separately. A mouse model of septic shock was induced using 12.5 mg / kg LPS.

[0083] 2. Experimental Methods

[0084] 1) Construction of a mouse model of septic shock

[0085] Female 7-week-old C57BL / 6J mice, acclimatized for one week, were evenly distributed into various groups based on body weight and recorded by ear tagging. Grouping was as follows: LPS group, LPS+DXM (5 mg / kg) group, LPS+DHZD (10 mg / kg, L) group, LPS+DHZD (20 mg / kg, M) group, LPS+DHZD (40 mg / kg, H) group, and LPS+DXM+DHZD (L) group; a total of 6 groups, 10 mice per group. On the day of model establishment, LPS was diluted to the experimental concentration with sterile PBS, and the optimal lethal concentration of 12.5 mg / kg was determined in preliminary experiments and administered intraperitoneally to establish a mouse septic shock model. The mice were observed for 180 hours to assess their condition and survival.

[0086] 3. Measurement and Statistical Methods

[0087] The mice were observed for 7 consecutive days and their survival status was recorded. Survival curves were plotted using Graphpad Prism 8 software, and survival analysis was performed using the Log-Rank test.

[0088] 4. Experimental Results

[0089] Experimental results are as follows Figure 4 As shown: ① LPS at a dose of 12.5 mg / kg successfully induced a mouse model of septic shock. All mice in the model group died within 40 hours, and the mice exhibited symptoms such as lethargy, shivering, piloerection, and chills during the experiment; ② The positive control drug DXM effectively protected mice with septic shock, with a survival rate of 60%; ③ DHZD alone at 10 mg / kg showed a protective trend, with a survival rate of 20%; ④ The survival rates of DHZD alone at 20 mg / kg and 40 mg / kg were consistent, both at 30%, which was statistically significant compared to the LPS model group; ⑤ The combination of 10 mg / kg DHZD and DXM provided 100% protection to mice with septic shock, and the protective effect was superior to that of DXM alone, which was statistically significant.

[0090] These results indicate that DHZD has a certain protective effect on LPS-induced septic shock model mice and can prolong the survival of mice. The combination of DHZD and DXM has a better synergistic protective effect.

[0091] (5) DHZD reduces LPS-induced secretion of cytokines in mouse serum.

[0092] 1. Sample preparation

[0093] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 80 mg / mL, and further diluted with sterile PBS to a concentration of 4 mg / mL to prepare DXM solution. An acute peritonitis mouse model was induced using 10 mg / kg LPS.

[0094] 2. Experimental Methods

[0095] 1) Preparation of a mouse model of acute peritonitis

[0096] Female 7-week-old C57BL / 6J mice, acclimatized for one week, were evenly distributed into different groups based on their body weight and recorded by ear tagging. Grouping was as follows: PBS (0.2 mL / mouse), LPS (10 mg / kg), LPS+DXM (5 mg / kg), LPS+DHZD (40 mg / kg), and LPS+DXM (5 mg / kg)+DHZD (10 mg / kg); a total of 5 groups, 9 mice per group. On the day of modeling, LPS was diluted to the experimental concentration with sterile PBS and injected intraperitoneally at 10 mg / kg to establish an acute peritonitis model in mice. Simultaneously, DHZD and / or DXM were injected intraperitoneally, and samples were collected 12 hours later.

[0097] 2) Preparation of mouse serum samples

[0098] Holding the mouse's head and face skin firmly with the index finger and thumb of your left hand to fully expose the eyeballs, place the mouse head down with its eyeballs aligned with the open 1.5mL Epp tube. Quickly grasp the eyeballs with ophthalmic curved forceps and pull them outwards to collect all the blood into the 1.5mL Epp tube, avoiding blood droplets adhering to the tube walls and causing hemolysis. Incubate at 4°C for 3 hours, then centrifuge (4°C, 3500rpm × 25min), and collect the clear, transparent serum supernatant into a new 1.5mL Epp tube.

[0099] 3. Measurement and Statistical Methods

[0100] The secretion of cytokines in mouse serum was detected by ELISA. The results of each group were statistically analyzed using a t-test, and the data were expressed as mean values. ± standard deviation (SD) is expressed.

[0101] 4. Experimental Results

[0102] Experimental results are as follows Figure 5As shown: ① The levels of IL-6, TNF-α, MCP-1, and IL-10 in mice with LPS-induced acute peritonitis were significantly increased, indicating that infection promotes the production of inflammatory and chemokine factors; ② Both the positive control drug DXM and DHZD alone inhibited the production of IL-6, TNF-α, MCP-1, and IL-10, which was statistically significant; ③ The combined administration of DHZD and DXM significantly reduced the production of IL-6, TNF-α, MCP-1, and IL-10, with the inhibitory effect on IL-6 being more significant than that of the positive control drug DXM alone, which was statistically significant.

[0103] These results indicate that, compared with DXM monotherapy, combination therapy can better inhibit the production of important inflammatory factors.

[0104] (6) DHZD has a protective effect against body temperature in CRKP-infected mice.

[0105] 1. Sample preparation

[0106] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 80 mg / mL, and then further diluted to 4 mg / mL with sterile PBS. Levofloxacin (LVX) solution and ineffective antibiotic MEM solution were also prepared. A mouse model of septic shock was prepared using a lethal dose of CRKP.

[0107] 2. Experimental Methods

[0108] 1) Construction of a mouse model of septic shock

[0109] Female 7-week-old C57BL / 6J mice that had been acclimatized for one week were evenly distributed into different groups based on their body weight, and ear tags were used for recording. Grouping: PBS group, CRKP group (9×10⁻⁶) 7 The mice were divided into seven groups: CFU / mouse, CRKP+MEM (10 mg / kg), CRKP+LVX (75 mg / kg), CRKP+DHZD-L (20 mg / kg), CRKP+DHZD-H (40 mg / kg), and CRKP+MEM+DHZD-L, with 10 mice per group. On the day of modeling, the prepared CRKP bacterial solution was diluted to the experimental concentration with sterile PBS and injected intraperitoneally to establish a mouse septic shock model. The drugs were injected simultaneously. The mice's living conditions and survival were observed continuously for 150 hours.

[0110] 3. Measurement and Statistical Methods

[0111] The survival status and condition of mice in each group were continuously observed and recorded. Survival curves were plotted using Graphpad Prism 8 software, and survival analysis was performed using the Log-Rank test. The body temperature of the mice was recorded using an infrared thermometer before modeling and 9 hours later.

[0112] 4. Experimental Results

[0113] Experimental results are as follows Figure 6 As shown: ① CRKP successfully induced a mouse model of septic shock, with a mortality rate of 90% within 40 hours in the model group, and mice exhibiting symptoms such as lethargy, shivering, piloerection, and chills during the experiment; ② The positive control drug LVX effectively protected mice with septic shock, with a survival rate of 70%; ③ The ineffective antibiotic MEM had no protective effect against CRKP-induced sepsis; ④ DHZD did not increase the survival rate of mice infected with lethal doses of CRKP; ⑤ DHZD could slow the drop in body temperature in septic shock model mice infected with CRKP, which was beneficial for maintaining or restoring normal body temperature.

[0114] This part of the results indicates that DHZD has a certain thermoprotective effect on the septic shock model mice constructed by infecting CRKP.

[0115] (7) DHZD has a protective effect on organs in CRKP-infected mice.

[0116] 1. Sample preparation

[0117] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 80 mg / mL, and further diluted with sterile PBS to a concentration of 4 mg / mL to prepare LVX solution; 6 × 10 7 A sepsis mouse model was established using CFU / mouse of CRKP.

[0118] 2. Experimental Methods

[0119] 1) Construction of a mouse sepsis model

[0120] Female 7-week-old C57BL / 6J mice, which had been acclimatized for one week, were evenly distributed into different groups based on their body weight, and ear tags were used for recording. Grouping: PBS group, CRKP group (6×10⁻⁶). 7 Four groups were established, with 10 mice per group: CFU / mouse, CRKP+LVX (75 mg / kg), and CRKP+DHZD-H (40 mg / kg). On the day of modeling, the prepared CRKP bacterial solution was diluted with sterile PBS to the experimental concentration and injected intraperitoneally to establish a mouse sepsis model. The drugs were injected simultaneously, and samples were collected 12 hours later.

[0121] 3. Measurement and Statistical Methods

[0122] Mice were euthanized, and their lungs and livers were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE). Histopathological changes were observed using a Pannoramic MIDI II microscope. Pulmonary edema, alveolar infiltration, and pulmonary vasculitis were scored using a 12-point pathological scale. Statistical analysis of the results was performed using a t-test, and data were expressed as mean values. ± standard deviation (SD) is expressed.

[0123] 4. Experimental Results

[0124] Experimental results are as follows Figure 7 As shown: Compared with the PBS group, the CRKP group mice showed obvious inflammatory cell infiltration in the lung tissue, a large amount of fluid exudation in the alveoli, and thickening of the pulmonary interstitium; the liver showed inflammatory cell infiltration, hepatocyte swelling and necrosis, partial nucleus fragmentation and dissolution, and destruction of the liver plate structure. DHZD can alleviate lung and liver tissue damage. Lung tissue pathological scores (pulmonary edema, alveolar infiltration, and pulmonary vasculitis) showed that DHZD has a protective effect against histopathological damage.

[0125] These results indicate that DHZD has a protective effect on the lungs and liver of mice with a sepsis model constructed by infecting CRKP.

[0126] (8) DHZD promotes the phagocytosis of pHrodo-labeled E. coli by Raw264.7 cells.

[0127] 1. Sample preparation

[0128] Dihydroquinone C was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg / mL, further diluted with PBS (phosphate buffered saline) to a stock solution of 40 mg / mL, and further diluted with serum-free DMEM cell culture medium to a sample solution of 15 μM. Lipopolysaccharide (LPS) of 100 ng / mL was used as the stimulant for cell-level experiments.

[0129] 2. Experimental Methods

[0130] Perform the macrophage phagocytic function test as follows:

[0131] Raw264.7 cells 1×10 5 100 μL of cells were seeded into 96-well cell culture plates and cultured overnight.

[0132] ②After 24 hours, add fresh culture medium containing LPS (100 ng / mL) alone, or add fresh culture medium containing LPS (100 ng / mL) and DHZD (15 μM);

[0133] ③ After 24 hours, add 10 μL of pHrodo fluorescently labeled E. coli lyophilized powder (pre-ultrasonic vibration at 37°C for 15 min), and incubate at 37°C in a CO2-free incubator in the dark for 1 hour.

[0134] ④ Discard the culture medium, gently rinse each well of the 96-well plate three times with sterile PBS, resuspend each well of the cells in 2% BSA solution, and then detect the phagocytosis of pHrodo fluorescently labeled E. coli by Raw264.7 cells using FACS technology.

[0135] 3. Measurement and Statistical Methods

[0136] Flow cytometry was used to detect the phagocytic function of macrophages under DHZD intervention. The results of each group were statistically analyzed using a t-test, and the data were expressed as mean values. ± standard deviation (SD) is expressed.

[0137] 4. Experimental Results

[0138] Experimental results are as follows Figure 8 As shown, DHZD significantly increased the number of pHrodo fluorescently labeled E. coli phagocytosed by Raw264.7, suggesting that it can promote the phagocytosis of bacteria by mouse macrophages.

[0139] This part of the results indicates that DHZD can promote phagocytosis by macrophages.

[0140] (II) Formulation

[0141] (1) Preparation of dihydroquinoline C tablets

[0142] After mixing 10g of dihydroquinone C with 87.5g of excipients (white quinone: lactose = 7:3, mass ratio), 95% ethanol was added for granulation, drying, granulation (sieving), and 2.5g of sodium stearate was added and mixed evenly before tableting. Each tablet weighed 200mg, of which the content of dihydroquinone C was 10mg.

[0143] (2) Preparation of dihydroquinone C powder for injection

[0144] Dissolve 1g of dihydroquinone C and 5g of mannitol in 170mL of water for injection. After initial mixing, bring the volume to 200mL. Filter the solution and fill 1mL into vials. Freeze-dry, seal, and sterilize to obtain a lyophilized powder injection containing 5mg of dihydroquinone C per vial.

[0145] (3) Preparation of dihydroquinone C capsules

[0146] After mixing 15g of dihydroquinone C with 135g of excipients (white quinone: lactose = 7:3, mass ratio), 95% ethanol was added to granulate, dried, granulated (sieved), and filled into capsules, each weighing 150mg, of which the content of dihydroquinone C was 15mg.

[0147] In summary, dihydroquinone C significantly inhibited the production of cytokines (IL-6, IL-1β, TNF-α, IFN-β, MCP-1, and IL-10) in mouse macrophages (Raw264.7) induced by LPS, particularly inhibiting the production of these cytokines in the serum of mice in a sepsis model. It also improved the survival rate of mice in the sepsis model and exerted protective effects on body temperature and organs. Furthermore, dihydroquinone C inhibited the expression of co-stimulatory molecules (CD80, CD86, and CD40) and MHC class II molecules (Iab) on the surface of dendritic cells, thereby suppressing T cell activation, reducing cytokine storms, and promoting macrophage phagocytosis of bacteria. Therefore, dihydroquinone C can be used as an active ingredient in the preparation of anti-inflammatory and immunomodulatory drugs, especially for the treatment of sepsis, thereby alleviating the difficult-to-control critical symptoms in clinical treatment of sepsis and demonstrating significant clinical application value.

Claims

1. Use of the combination of dihydromethionine C and dexamethasone in the preparation of a drug for sepsis.

2. The use according to claim 1, characterized in that: The drug improves the survival rate of sepsis.

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