CN117338766A - Application of mejunin C in dihydro - Google Patents
Application of mejunin C in dihydro Download PDFInfo
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- CN117338766A CN117338766A CN202311424428.4A CN202311424428A CN117338766A CN 117338766 A CN117338766 A CN 117338766A CN 202311424428 A CN202311424428 A CN 202311424428A CN 117338766 A CN117338766 A CN 117338766A
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- dhzd
- dihydro
- inflammatory
- sepsis
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- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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Abstract
The invention discloses an application of mevalonate C in dihydro, which refers to the application of one or more of mevalonate C in dihydro or pharmaceutically acceptable salts thereof and the like as an active ingredient in preparing anti-inflammatory drugs, and the drugs have an immunoregulatory effect. Experimental results show that the mejunin C in the dihydro can reduce the excessive immune response of the organism to infection or induction, relieve the critical symptoms which are difficult to control in the sepsis treatment, and has medicinal value.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to anti-inflammatory and immunoregulatory application of mevalonate C in dihydro.
Background
The occurrence of excessive inflammatory reactions is increasingly considered a critical factor in sepsis mortality, particularly after discharge from hospital. According to the third international consensus definition of sepsis and septic shock, sepsis is a life threatening organ dysfunction due to a deregulation of the host's response to infection, a clinical disease with complex immunopathophysiology during which inflammation and immunosuppression may occur sequentially or simultaneously. Early in the systemic inflammatory response, the immune system can rapidly restore immune balance if it eliminates pathogens in time. If pathogens are not cleared in time, an imbalance in immunomodulation results. In this case, the patient is susceptible to secondary infections, resulting in long-term immunosuppression, immune failure, and even physical disability, also known as persistent inflammatory immunosuppression catabolic syndrome, which is also common in critical diseases caused by other non-infectious lesions, such as secondary major wounds, pancreatitis, and the like.
Since excessive inflammatory reactions and immunosuppression do not occur independently, they often coexist during the pathological course of sepsis. Thus, sepsis treatment is troublesome, being one of the most common causes of death in hospitalization and Intensive Care Units (ICU), and also one of the major causes of long-term death in severely wounded patients, with a first year mortality rate of 15% after discharge from hospital for sepsis survivors, and a subsequent 5 year mortality rate of 6.8%. In recent years, therapeutic approaches combining anti-inflammatory and immunomodulation have become a focus of research. Immunomodulatory treatments such as Ulinastatin (UTI) and tα1 can improve organ function and reduce mortality in severe sepsis patients. However, the combined administration mode, optimal dosage, treatment course and the like of the medicines still need to be further confirmed by clinical researches. In addition, the excessive immune response generated by the body in the case of severe infection is also an important factor in causing high mortality rate of sepsis, and thus control of the excessive immune response is also an effective direction of treatment.
The dihydromevalanin C (3-epi-Zaluzanin C, isozalutin C, DHZD) is a sesquiterpenoid (Li, C.; yu, X.; lei, X. "A Biomimetic Total Synthesis of (+) -Ainsliaadimer A" org. Lett.2010,12, 4284-4287.), which contains allyl alcohol structural groups (as shown in Compound 1 below) without a conjugated system between the corresponding hydroxyl groups and double bonds, and in the dehydromevalanin C (as shown in Compound 2 below) has alpha, beta-ketone structural groups which belong to the conjugated system. There is no report on the use of mevalonate C in dihydro as an active ingredient of anti-inflammatory and immunomodulating drugs.
Dehydro-mejunin C, although having some anti-inflammatory, antibacterial effect (chinese patent CN105640937A, CN112587517 a), is limited by its cytotoxicity and has not been studied intensively in animal experiments, for example, against severe sepsis models.
Disclosure of Invention
The invention provides an application of mevalonate C in dihydro, which aims to develop the medicinal value of mevalonate C in dihydro in relieving critical symptoms which are difficult to control in clinical treatment of sepsis and other diseases.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in an animal model for simulating sepsis, experiments show that the mevalonate C (DHZD) in dihydro can play an obvious role in protecting the body temperature and organs of mice in a sepsis shock and sepsis model of mice infected with carbapenem-resistant klebsiella pneumoniae (CRKP); DHZD can improve survival in a lethal dose of gram-negative bacterial Lipopolysaccharide (LPS) -induced mouse model of septic shock, and intervention with DHZD can down-regulate the expression levels of inflammatory and chemokines (such as IL-6, TNF- α, IL-10 and MCP-1) in serum of septic mice.
Among the important innate immune cells (monocytes/macrophages, dendritic cells) involved in inflammatory response, DHZD has been found experimentally to inhibit the activation of PI3K/Akt/p70S6K signaling pathway, thereby down-regulating the expression levels of cytokines such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF- α), interleukin 1 beta (IL-1 beta), chemokines (e.g. MCP-1), interferon beta (IFN-beta), interleukin 10 (IL-10), etc.
The experimental results show that DHZD can inhibit excessive inflammatory response induced by LPS, so that the damage and even death of organisms caused by the excessive inflammatory response can be reduced. In the treatment of sepsis caused by CRKP infection, DHZD can reduce the excessive immune response of the body to pathogenic bacteria infection by regulating and controlling host immune response, and relieve the critical symptoms of sepsis which are difficult to control. Therefore, the application of the dihydromevalonate C in the invention specifically refers to the application of one or more of the dihydromevalonate C, stereoisomers, precursor compounds and pharmaceutically acceptable salts thereof as active ingredients with immunoregulatory action in preparing anti-inflammatory drugs, in particular to the preparation of drugs for achieving the anti-inflammatory purpose by combining immunoregulation.
Preferably, the active ingredient with immunoregulatory effect (e.g. mevalonate C) is administered alone or in combination with one or more of an antibiotic selected from one or more of carbapenem antibiotics, quinolone antibiotics, such as meropenem, levofloxacin, and a hormone selected from the clinically usual glucocorticoids dexamethasone.
Preferably, the medicament contains a small amount of minor ingredients and/or pharmaceutically acceptable carriers which do not affect the active ingredients, in addition to the active ingredients (e.g., active ingredients having an immunoregulatory effect), for example, sweeteners may be contained in the medicament to improve taste, antioxidants to prevent oxidation, and auxiliary materials necessary for various preparations, etc.
The dosage form of the drug is not limited as long as the active ingredient can reach the body, and for example, the dosage form of the drug is a tablet, a capsule, a powder, a granule, a dripping pill, a syrup, a solution, a suspension, an injection, a tincture, an oral liquid, an aerosol, a buccal agent, a granule, a pill, a powder and other common dosage forms or a nanometer preparation and other sustained-release dosage forms.
In the above technical scheme, the "pharmaceutically acceptable salt" refers to a salt formed by mevalonate C in dihydro and a pharmaceutically acceptable inorganic acid or organic acid, wherein 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-naphthalene disulfonic acid, sub-fine acrylic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic 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 acid; by "pharmaceutically acceptable" is meant suitable for use in humans without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
In the above technical solutions, the "stereoisomer" refers to an isomer produced by a different arrangement of atoms in a molecule in space, for example: cis-trans isomers, enantiomers, conformational isomers, and the like.
In the above technical scheme, the "precursor compound" refers to a compound which is inactive in vitro but can be converted into dihydromevalonate C by metabolism or chemical reaction in an organism, thereby exerting the pharmacological action thereof.
The beneficial effects of the invention are as follows:
experiments show that the mejunin C in dihydro can reduce the excessive inflammatory reaction of organisms on pathogenic bacteria infection and endotoxin induction, exert body temperature and organ protection effects on sepsis, and improve the survival rate of severe sepsis. Experimental results show that the mejunin C in dihydro can be used as an active ingredient in anti-inflammatory and immunoregulatory medicines (including single administration or enhanced curative effect by combined administration with antibiotics and hormones), has important medicinal value, and provides important reference for searching novel candidate medicines and exploring more effective treatment strategies.
Drawings
FIG. 1 shows the cell viability (A) measured at a wavelength of 450nm after co-incubation (12, 24, 48 and 72 h) of DHZD and Raw264.7 cells at different concentrations, and Raw264.7 cells (2X 10) 5 cells/well) assayed secretion of IL-6 (B), TNF- α (C), MCP-1 (D), IFN- β (E), IL-1β (F), IL-10 (G) in cell culture supernatants at different concentrations of DHZD (0, 3, 5, 10 and 15 μm) and LPS (100 ng/mL) at different times (6 and 18 h) by ELISA (medium is a negative control for cultured cells only, each stimulation experiment was repeated three times, i.e., n=3; * P, p<0.05；**,p<0.01；***,p<0.001)。
FIG. 2 shows a primary peritoneal macrophage (3.5X10) 5 cell/300. Mu.L) was plated on 24 well plates the next day after stimulation with different concentrations of DHZD (0, 3, 5, 10 and 15. Mu.M) and LPS (100 ng/mL) and cell culture supernatants were collected at different times (6 and 18 h) to examine IL-6 (A), TNF- α (B), MCP-1 (C), IL-1β (D), IFN- β (E), IL-10 (F) secretion and NO (G) (medium was a negative control for cultured cells only, each experiment was repeated three times, i.e., n=3; * P, p<0.05；**,p<0.01；***,p<0.001)。
FIG. 3 shows BMDCs (at 3.5X10) 5 cell/300. Mu.L density) was plated on 24-well plates and expression of costimulatory molecules CD40 (D), CD86 (E), CD80 (F) and MHC II (Iab) (G) on the surface of BMDCs was examined by flow cytometry after 6 and 18h stimulation with different concentrations of DHZD (0, 3, 5, 10 and 15. Mu.M) and LPS (100 ng/mL) and after 24h stimulation with DHZD (15. Mu.M) and LPS (100 ng/mL) in the collected cell culture supernatants and data analysis was performed by flow-through FloJo software; medium was a negative control for cultured cells only, and each experiment was repeated 3 times, i.e., n=3; * P, p<0.05；**,p<0.01；***,p<0.001；****,p<0.0001)。
Figure 4 shows the change in survival rate of female C57BL/6 mice (randomized into 6 groups of 10) following i.p. 12.5mg/kg of LPS per mouse with DHZD alone or in combination with DXM (p <0.05; p <0.01; p <0.001; p < 0.0001).
FIG. 5 shows the secretion of IL-6 (A), TNF-. Alpha. (B), MCP-1 (C) and IL-10 (D) in serum (serum prepared from mouse eyeball blood) by ELISA 12h after drug injection in PBS group, LPS (10 mg/kg) group, LPS+DXM (5 mg/kg) group, LPS+DHZD (40 mg/kg) group, LPS+DXM (5 mg/kg) +DHZD (10 mg/kg) group female C57BL/6 mice (randomized into 5 groups, 9 mice each).
FIG. 6 shows PBS group, CRKP (9×10) 7 CFU/group only), 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 female C57BL/6 mice (randomly divided into 7 groups, 10 mice per group) were subjected to intraperitoneal injection of lethal amounts of CRKP, and the state of the mice was observed every 2 to 3 hours up to 150 hours (A), and body temperature monitoring and changes (B and C) before and after molding were compared (<0.05；**,p<0.01；***,p<0.001；****,p<0.0001)。
FIG. 7 shows PBS group, CRKP (6×10) 7 CFU/group only), CRKP+LVX (75 mg/kg) group and CRKP+DHZD-H (40 mg/kg) group female C57BL/6 mice (randomly divided into 4 groups of 10 mice each) were intraperitoneally injected with 6X 10 7 CFUCRKP and after 12h mice were fixed with HE staining (a) for Lung (Lung), liver (Liver), and results of scoring (B) pulmonary edema, alveolar infiltration, and pulmonary vasculitis according to a 12 score pathology scale (x, p)<0.001；****,p<0.0001)。
FIG. 8 shows Raw264.7 cells (at 1X 10) 5 cell/100 μl density) and incubated with the ph rodo labeled e.coli in the absence of light for 24h after DHZD (15 μΜ) and LPS (100 ng/mL) stimulation, and flow assay (a) using FlowJo to analyze the flow results (B, C), wherein each experiment was repeated 3 times, i.e. n=3; * P is:<0.01；****,p<0.0001。
Detailed Description
The invention is further elucidated below in connection with the drawings and the embodiments. It should be understood that the examples are only for the purpose of illustrating the invention and are not intended to limit the scope of the invention.
Pharmacological experiments
(1) In vitro anti-inflammatory Activity of DHZD on Raw264.7 cells
1. Sample preparation
Methrin C (DHZD) in dihydro was dissolved in dimethyl sulfoxide (DMSO) to 100mg/mL and further diluted with PBS (phosphate buffered saline) to 40mg/mL of stock solution. Further diluting with serum-free DMEM cell culture medium to obtain sample solutions with gradient concentration of 3, 5, 10 and 15 μm (0 μm sample solution without DHZD is prepared); lipopolysaccharide (LPS) at 100ng/mL was used as a stimulator of the cell level experiments.
2. Experimental method
1) Culture of mouse macrophage cell line Raw264.7
Subculturing with DMEM high sugar medium containing 10% FBS at 37deg.C and 5% CO 2 Culturing in incubator. When the pooling rate reaches 70% -80% under the light microscope, the old culture medium is sucked and abandoned, 3mL/10mm dish fresh complete culture medium is added, adherent cells are scraped, cell mass is gently blown away, and then cell passage is carried out by proportionally retaining liquid, or the next experiment is carried out by plating after counting.
2) Cell proliferation/toxicity assay
After co-incubation of DHZD and raw264.7 cells at different concentrations, 10 μl of CCK8 enhanced solution was added per well according to the experimental procedure of the CCK-8 kit instructions, and after the addition of the reagents, the plates were gently rocked to aid in mixing, incubated in the incubator for 2h, and absorbance at 450nm was measured with an microplate reader. The cell viability was calculated as follows:
cell viability = [ (As-Ab)/(Ac-Ab) ] ×100%
Wherein As is absorbance of a drug experimental hole; ac is the absorbance of the negative control wells; ab is blank well absorbance.
3) Enzyme-linked immunosorbent assay (ELISA)
Dissolving the standard substance and capturing according to the procedure of the instruction of the kitAntibody and detection antibody, marking dilution times, split charging and storing in a low temperature refrigerator at-20 ℃. (1) Diluting a Capture antibody (Capture antibody) to a working concentration by using sterile PBS according to the number of samples and the total number of standard substances, adding a continuous sample gun into the plate, coating an ELISA plate at 100 mu L/hole, and standing at room temperature overnight; (2) the capture antibody is discarded the next day, 300 mu L of washing liquid (Wash buffer, PBS containing 0.05% Tween20, pH 7.2-7.4) is added into each hole by a row gun, the washing liquid is beaten dry on the paperboard after 1min, the washing liquid is washed for 3 times, and no residual washing liquid is ensured in the holes after the last time; (3) adding 300 mu L/well of Reagent diluent (PBS pH 7.2-7.4 of 1% BSA), attaching a sealing plate film, and sealing for 1h at room temperature; (4) discarding the reagent diluent, and repeating the plate washing operation (2)0 step; (5) respectively adding a sample to be detected (cell culture supernatant, properly diluting) and a standard substance of 100 mu L/hole, attaching a sealing plate film, and incubating for 2 hours at room temperature; (6) discarding the sample to be tested and the standard substance, and repeating the plate washing operation (2)3 step; (7) diluting the detection antibody (Detection antibody) to a working concentration by using a reagent diluent, adding the reagent diluent according to 100 mu L/hole, and incubating for 2 hours at room temperature; (8) discarding the detection antibody, and repeating the plate washing operation in the step (2); (2) 1 adding 100 mu L/hole of coupled horseradish peroxidase (HRP) diluted to a working concentration by a diluent, and incubating for 20min at room temperature in a dark place; (2) discarding HRP, and repeating the plate washing operation in step (2);adding 100 mu L/hole of a reaction substrate solution (Substrate solution) prepared in a ratio of 1:1, and reacting for 20min at room temperature in a dark place; />50. Mu.L/well of Stop solution (Stop solution) was added, and the OD at 450nm (detection wavelength)/570 nm (correction wavelength) was measured by an ELISA reader, and the corresponding protein concentration was calculated from the standard curve.
3. Method for measuring and counting
Using CCK-8 to detect drug toxicity to cells, ELISA detects cytokine secretion in the supernatant. The results of each group were statistically analyzed using T-test, and the data were averagedStandard Deviation (SD) is indicated.
4. Experimental results
The experimental results are shown in fig. 1: in Raw264.7 cells, no cytotoxicity of 0-15 mu M DHZD is observed within 72h, and normal growth of the cells is not affected. Furthermore, intervention with DHZD inhibits secretion of IL-6, TNF-alpha, MCP-1, IFN-beta, IL-1 beta and IL-10 by LPS stimulation and exhibits a clear dose-dependent relationship.
The results in this section indicate that: the mejunin C in dihydro can obviously inhibit the inflammatory factor and chemokine level of LPS induced Raw264.7, and has anti-inflammatory regulation effect.
(2) DHZD inhibits LPS-induced cytokine secretion in primary peritoneal macrophages of mice
1. Sample preparation
Methrin C in dihydro was dissolved in dimethyl sulfoxide (DMSO) to 100mg/mL and further diluted with PBS (phosphate buffered saline) to 40mg/mL of stock solution. Further diluting with serum-free DMEM cell culture medium to obtain sample solutions with gradient concentration of 3, 5, 10 and 15 μm (0 μm sample solution without DHZD is prepared); lipopolysaccharide (LPS) at 100ng/mL was used as a stimulator of the cell level experiments.
2. Experimental method
1) Induction and culture of Primary peritoneal macrophages (pM phi) in mice
SPF-grade C57BL/6J female mice (6-8 weeks old) were intraperitoneally injected with sterile 3.5% sodium thioglycolate solution, 1 mL/each of the left and right abdomen. After 3.5 days, mice were sacrificed by cervical dislocation and fully soaked in 75% ethanol for 5min for sterilization. The abdominal skin of the mice was cut off, the abdominal cavity was rinsed twice with a 20mL syringe that had been aspirated into a pre-warmed sterile DMEM, the rinse was collected in a sterile 50mL centrifuge tube, repeatedly aspirated and blown, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cell pellet was resuspended in DMEM medium containing 1% diabody and 10% fbs, blown off with a 10mL pipette, diluted and plated after counting. After conventional culture for 2h, the liquid is changed, and the adherent cells are pMphi.
LPS (100 ng/mL) and DHZD (0-15 mu M) with different concentrations are simultaneously added into pM phi cell culture medium, and after different time points of action, cell culture supernatants are collected.
2) Determination of nitric oxide content by Griess method
The procedure was according to the kit instructions: (1) taking out Griess Reagent I and II and standard substances, thawing the Griess Reagent I and II and recovering the Griess Reagent I and II to room temperature; (2) diluting the standard (1-100 mu M) with a cell culture solution; (3) each standard and sample (cell culture supernatant) was added to a 96-well flat bottom plate at 50 μl/well; (4) adding Griess Reagent I Reagent into each well at a concentration of 50 μl/well; (5) adding Griess Reagent II reagent to each well at 50 μl/well; (6) mixing the culture plates evenly, and then measuring absorbance at 540 nm; (7) the concentration of Nitric Oxide (NO) in the sample was calculated from the standard curve.
3. Method for measuring and counting
Respectively ELISA method and Griess method, detecting secretion of inflammatory factor, chemokine and NO in culture supernatant of each well cell, performing statistical analysis of each experimental result by T test, and averaging dataStandard Deviation (SD) is indicated.
4. Experimental results
The experimental results are shown in fig. 2: in the primary peritoneal macrophages of mice, DHZD has anti-inflammatory activity, can inhibit secretion of LPS-induced pro-inflammatory cytokines IL-6, TNF-alpha, IL-1 beta, type I interferon IFN-beta and NO, and reduce secretion of chemokines MCP-1 and anti-inflammatory cytokines IL-10.
The results in this section indicate that: DHZD down regulates LPS-induced inflammatory responses in macrophages by reducing pro-inflammatory cytokine and chemokine production, suggesting that DHZD exhibits good anti-inflammatory activity at the cellular level.
(3) DHZD inhibits secretion of inflammatory factors in LPS-induced dendritic cells and reduces expression of cell surface costimulatory molecules
1. Sample preparation
Methrin C in dihydro was dissolved in dimethyl sulfoxide (DMSO) to 100mg/mL and further diluted with PBS (phosphate buffered saline) to 40mg/mL of stock solution. Further diluting with serum-free DMEM cell culture medium to obtain sample solutions with gradient concentration of 3, 5, 10 and 15 μm (0 μm sample solution without DHZD is prepared); lipopolysaccharide (LPS) at 100ng/mL was used as a stimulator of the cell level experiments.
2. Experimental method
1) Induction and culture of dendritic cells (BMDC) derived from mouse bone marrow
Taking SPF grade male C57BL/6J mice (4 weeks old) with 4 bones in two hind limbs, sucking pre-warmed 1640 with a 1mL syringe, repeatedly flushing bone marrow from two ends of the bones until the bones become white, blowing off the bone marrow with the syringe in a culture medium, centrifuging (1000 rpm×5min, RT), discarding supernatant, flicking cell mass, adding 2 mL/red-breaking liquid, breaking red at room temperature for 2.5min, adding equal volume of 1640 containing serum to stop breaking red, centrifuging (1000 rpm×5min, RT), discarding supernatant, flicking cell mass gently, adding 21 mL/DC complete medium (10%FBS1640+50 ng/mL mGM-CSF+4ng/mL mIL-4), re-suspending, adding into six-well plate after complete mixing, adding 2.5 mL/well, and adding CO at 37 ℃ and 5% 2 Culturing in incubator, supplementing DC complete culture medium after 3 days, and counting and plating after 5 days.
2) Flow cytometry (FACS) detection of costimulatory molecule expression
(1) The induced mouse bone marrow-derived dendritic cells were prepared into single cell suspensions, and 12-well plates (5X 10) 5 Each well, 800. Mu.L) was added with LPS (100 ng/mL) and the drug DHZD (15. Mu.M), respectively;
(2) after 24h of co-incubation, cells from each well of the 12-well plate were gently blown down and transferred to sterile 1.5mL Epp tubes;
(3) centrifuging at a low temperature at 1400rpm for 5min;
(4) discarding the supernatant, and centrifuging the discarded supernatant after resuspension of 250 mu L of PBS;
(5) preparing dead living cell dye solution (PBS, zombie, fcR) according to the instruction proportion, 50 mu L/tube, gently mixing cells (RT, light-shielding) for 10min;
(6) washing for one time: add Fc Buffer 220. Mu.L/tube and centrifuge at 4 ℃ (1400 rpm X5 min);
(7) discarding the supernatant, reversely buckling paper towel, adding 50 mu L of antibody working solution, fully oscillating and uniformly mixing, keeping out of the sun at 4 ℃, and incubating for 25min;
(8) washing for one time: add Fc Buffer 200. Mu.L/tube and centrifuge at 4 ℃ (1400 rpm X5 min); after centrifugation, the supernatant was discarded, the paper towel was inverted, 200. Mu.L of PBS was added, and the mixture was thoroughly mixed by shaking to complete the collection of cells, and the preparation was performed.
3. Method for measuring and counting
Detecting secretion of inflammatory factors in culture supernatant of each well by ELISA method, detecting expression of BMDC surface costimulatory molecules and MHC class II molecules by flow, performing statistical analysis by T test on experimental results of each group, and averaging dataStandard Deviation (SD) is indicated.
4. Experimental results
The experimental results are shown in fig. 3: DHZD can reduce secretion of LPS-induced pro-inflammatory cytokines IL-6, TNF- α and IL-12p70, suggesting that DHZD can inhibit LPS-induced dendritic cells from secreting a large number of inflammatory factors; DHZD can significantly inhibit the expression of costimulatory molecules and MHC class II molecules, suggesting that DHZD can reduce the ability of dendritic cells to activate T cells.
(4) DHZD alone or in combination with dexamethasone can increase survival of LPS-induced sepsis mice
1. Sample preparation
Dissolving mevalonate C in dihydro with dimethyl sulfoxide (DMSO) to 80mg/mL, further diluting with sterile PBS to 4mg/mL, and preparing Dexamethasone (DXM) liquid medicine; a12.5 mg/kg LPS induced sepsis shock mouse model was used.
2. Experimental method
1) Construction of a mouse sepsis shock model
Female 7-week-old C57BL/6J mice, which had been adaptively bred for 1 week, were evenly distributed to each group according to body weight, and ear-marked. Grouping condition: LPS group, LPS+DXM (5 mg/kg), LPS+DHZD (10 mg/kg, L) group, LPS+DHZD (20 mg/kg, M) group, LPS+DHZD (40 mg/kg, H) group, LPS+DXM+DHZD (L) group; 6 groups, 10/group. On the day of modeling, LPS is diluted to an experimental concentration by sterile PBS, the optimal lethal concentration is 12.5mg/kg according to a pre-experimental fumbling, and the injection is performed intraperitoneally, so that a sepsis shock model of a mouse is constructed, and the medicines are injected simultaneously. The state of life and survival of the mice were continuously observed for 180 h.
3. Method for measuring and counting
Survival and status of each group of mice were continuously observed for 7 days and recorded, survival curves were drawn with Graphpad prism 8 software and survival analysis was performed using Log-Rank test.
4. Experimental results
The experimental results are shown in fig. 4: (1) the LPS with the dosage of 12.5mg/kg successfully induces a sepsis shock mouse model, mice in the model group die completely within 40 hours, and the mice show symptoms such as somnolence, chestnut, hair erection, coldness and the like in the experimental process; (2) the positive drug DXM can effectively protect sepsis shock mice, and the survival rate is 60%; (3) the trend of 10mg/kg of DHZD alone acting on organism protection is 20% survival; (4) the individual action survival rates of 20mg/kg and 40mg/kg of DHZD are 30%, and the DHZD has statistical significance compared with LPS model groups; (5) 10mg/kg DHZD combined with DXM can protect sepsis shock mice 100%, and the protection effect is better than that of a DXM single-use group, so that the method has statistical significance.
The results in this section indicate that: the DHZD has a certain protection effect on mice with the LPS-induced sepsis shock model, can prolong the survival time of the mice, and has better synergistic protection effect when being combined with the DXM.
(5) DHZD reduces LPS-induced cytokine secretion in mouse serum
1. Sample preparation
Dissolving mejunin C in dihydro with dimethyl sulfoxide (DMSO) to 80mg/mL, further diluting with sterile PBS to 4mg/mL, and preparing DXM liquid medicine; a10 mg/kg LPS-induced acute peritonitis mouse model was used.
2. Experimental method
1) Preparation of mouse acute peritonitis model
Female 7-week-old C57BL/6J mice, which had been adaptively bred for 1 week, were evenly distributed to each group according to body weight, and ear-marked. Grouping condition: PBS (0.2 mL/group), LPS (10 mg/kg), LPS+DXM (5 mg/kg), LPS+DHZD (40 mg/kg), LPS+DXM (5 mg/kg) +DHZD (10 mg/kg); totally 5 groups, 9 mice/group. On the day of modeling, diluting LPS to experimental concentration by using sterile PBS, and performing intraperitoneal injection according to 10mg/kg to construct an acute peritonitis model of the mice, and simultaneously, respectively performing intraperitoneal injection of DHZD and/or DXM drugs, and obtaining materials after 12 hours.
2) Preparation of mouse serum samples
The skin of the face of the mouse head is clamped by the index finger and the thumb of the left hand, the eyeballs are fully exposed, the mouse head faces downwards, the eyeballs are aligned to a 1.5mL Epp tube with an opening, the eyeballs are rapidly clamped by the ophthalmic curved forceps and then rapidly pulled outwards, and blood is completely collected in the 1.5mL Epp tube, so that blood drop wall hanging hemolysis is avoided. The supernatant clear and transparent serum was aspirated into a fresh 1.5mL Epp tube by standing in a refrigerator at 4deg.C for 3h and then centrifuging (4deg.C, 3500rpm X25 min).
3. Method for measuring and counting
ELISA method is used to detect the secretion of cytokines in serum of mice, each group of experimental results is statistically analyzed by T test, and the data are averagedStandard Deviation (SD) is indicated.
4. Experimental results
The experimental results are shown in fig. 5: (1) the LPS-induced levels of IL-6, TNF-alpha, MCP-1 and IL-10 in mice with acute peritonitis are significantly increased, i.e., infection promotes the production of inflammatory factors and chemokines; (2) the positive drug DXM and the single administration of DHZD inhibit the production of IL-6, TNF-alpha, MCP-1 and IL-10, and have statistical significance; (3) the combined administration of DHZD and DXM greatly reduces the production of IL-6, TNF-alpha, MCP-1 and IL-10, wherein the IL-6 has more obvious inhibition effect than the single positive drug DXM group, and has statistical significance.
The results in this section indicate that: the combination can better inhibit the production of important inflammatory factors than DXM alone.
(6) DHZD has protective effect on body temperature of CRKP infected mice
1. Sample preparation
Dissolving mevalonate C in dihydro with dimethyl sulfoxide (DMSO) to 80mg/mL, further diluting with sterile PBS to 4mg/mL, and preparing Levofloxacin (LVX) medicinal liquid and ineffective antibiotic MEM medicinal liquid; a lethal dose of CRKP was used to prepare a sepsis shock mouse model.
2. Experimental method
1) Construction of a mouse sepsis shock model
Female 7-week-old C57BL/6J mice, which had been adaptively bred for 1 week, were evenly distributed to each group according to body weight, and ear-marked. Grouping condition: PBS group, CRKP (9X 10) 7 CFU/group only), CRKP+MEM (10 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+DHZD-L group; a total of 7 groups, 10/group. On the day of modeling, the prepared CRKP bacterial liquid is diluted to experimental concentration by sterile PBS, and is injected intraperitoneally, so as to construct a sepsis shock model of a mouse, and the medicines are injected simultaneously. The state of life and survival of the 150h mice were continuously observed.
3. Method for measuring and counting
Survival and status of each group of mice were continuously observed and recorded, survival curves were drawn with Graphpad prism 8 software, and survival analysis was performed using Log-Rank test. Before and after the molding, the body temperature of the mice is recorded by an infrared thermometer.
4. Experimental results
The experimental results are shown in fig. 6: (1) CRKP successfully induces a sepsis shock mouse model, the death rate of a model group mouse reaches 90% within 40h, and the mice have symptoms of somnolence, chestnut, hair erection, coldness and the like in the experimental process; (2) the positive medicine LVX can effectively protect sepsis shock mice, and the survival rate is 70%; (3) the ineffective antibiotic MEM has no protective effect on CRKP-induced sepsis; (4) DHZD does not increase survival in lethal amounts of CRKP-infected mice; (5) DHZD can slow down the decline of body temperature in sepsis shock model mice after CRKP infection, and is beneficial to maintaining or restoring normothermia.
The results in this section indicate that: DHZD has a certain body temperature protective effect on sepsis shock model mice constructed by infection with CRKP.
(7) DHZD has protective effect on viscera of CRKP infected mice
1. Sample preparation
Dimethyl for mejunin C in dihydroDissolving sulfoxide (DMSO) to 80mg/mL, diluting with sterile PBS to 4mg/mL, and preparing LVX liquid medicine; using 6X 10 7 CFU/CRKP alone mice models of sepsis were prepared.
2. Experimental method
1) Construction of a mouse sepsis model
Female 7-week-old C57BL/6J mice, which had been adaptively bred for 1 week, were evenly distributed to each group according to body weight, and ear-marked. Grouping condition: PBS group, CRKP group (6×10) 7 CFU/alone), crkp+lvx (75 mg/kg) group and crkp+dhzd-H (40 mg/kg) group; there were 4 groups, 10/group. On the day of molding, the prepared CRKP bacterial liquid is diluted to experimental concentration by sterile PBS, and is injected intraperitoneally to construct a mouse sepsis model, and the medicines are injected simultaneously, and the materials are obtained after 12 hours.
3. Method for measuring and counting
Mice were sacrificed, lungs and livers of the mice were removed, fixed in 4% paraformaldehyde, paraffin embedded, and stained with HE and observed for histopathological changes using a PANNORAMIC MIDI II microscope. Pulmonary edema, alveolar infiltration, and pulmonary vasculitis were scored according to a 12-point pathology scale, each set of experimental results was statistically analyzed using T-test, and the data was averagedStandard Deviation (SD) is indicated.
4. Experimental results
The experimental results are shown in fig. 7: compared with PBS group, CRKP group mice lung tissue can be seen to be obvious in inflammatory cell infiltration, a large amount of liquid in alveoli is exuded, and lung interstitium is thickened; the liver can be infiltrated by inflammatory cells, the hepatocytes are swollen and necrotized, part of nuclei are disintegrated and dissolved, and the liver plate structure is damaged. DHZD can reduce lung and liver tissue damage. Lung tissue pathology scores (pulmonary oedema, alveolar infiltration and pulmonary vasculitis) show that DHZD has protective effects on tissue pathology lesions.
The results in this section indicate that: DHZD has protective effects on lung and liver of sepsis model mice constructed by infection with CRKP.
(8) DHZD promotes phagocytosis of pHrodo-labeled e.coli by raw264.7 cells
1. Sample preparation
Dissolving mevalonate C in dihydro into 100mg/mL with dimethyl sulfoxide (DMSO), diluting into 40mg/mL stock solution with PBS (phosphate buffer solution), and diluting into 15 μm sample solution with serum-free DMEM cell culture medium; lipopolysaccharide (LPS) at 100ng/mL was used as a stimulator of the cell level experiments.
2. Experimental method
The macrophage phagocytic function detection experiment was performed as follows:
raw264.7 cells 1×10 5 cells/100. Mu.L were plated in 96 well cell culture plates and incubated overnight;
(2) after 24h, fresh medium containing LPS (100 ng/mL) alone or fresh medium containing LPS (100 ng/mL) and DHZD (15. Mu.M) was added;
(3) after 24h, 10. Mu.L of pHrodo fluorescent-labeled E.coli (E.coli) lyophilized powder (ultrasonic vibration at 37 ℃ in advance for 15 min) was added, and no CO was present at 37 ℃ 2 Incubating for 1h in the incubator in a dark place;
(4) the medium was discarded, the wells in the 96-well plate were gently rinsed 3 times with sterile PBS, resuspended in 2% BSA solution, and then assayed for phagocytosis of pHrodo fluorescent-labeled E.coli by Raw264.7 cells by FACS techniques.
3. Method for measuring and counting
Detecting macrophage phagocytic bacteria function under DHZD intervention by flow cytometry, performing statistical analysis on experimental results of each group by adopting T test, and averaging dataStandard Deviation (SD) is indicated.
4. Experimental results
The experimental results are shown in fig. 8: DHZD can significantly increase the amount of raw264.7 phagocytosis of pHrodo fluorescent marker e.coli, suggesting that it can promote phagocytosis of bacteria by mouse macrophages.
The results in this section indicate that: DHZD promotes phagocytosis by macrophages.
(II) formulations
(1) Preparation of mejulin C tablets in dihydro
10g of mevalonate C in dihydro and 87.5g of auxiliary materials (white lake essence: lactose=7:3 in mass ratio) are mixed, then 95% of ethanol is added for granulating, drying, granulating (sieving), 2.5g of sodium stearate is added for uniformly mixing, and tabletting is carried out, wherein each tablet weighs 200mg, and the content of mevalonate C in dihydro is 10mg.
(2) Preparation of dihydromevalonate C powder injection
Dissolving 1g of mevalonate C in 170mL of water for injection, uniformly mixing for the first time, then fixing the volume to 200mL, filtering the obtained solution, filling into penicillin bottles, filling 1mL of the solution into each bottle, freeze-drying, sealing and sterilizing to obtain freeze-dried powder injection containing 5mg of mevalonate C in each bottle.
(3) Preparation of bifenthrin C capsule
15g of mevalonate C in dihydro and 135g of auxiliary materials (white lake essence: lactose=7:3 in mass ratio) are mixed, added with 95% of ethanol for granulating, dried, granulated (sieved) and filled into capsules, wherein the weight of each capsule is 150mg, and the content of mevalonate C in dihydro is 15mg.
In conclusion, the dihydromevalonate C can obviously inhibit the production of cytokines (IL-6, IL-1 beta, TNF-alpha, IFN-beta, MCP-1 and IL-10) of the mouse macrophage Raw264.7 under the induction of LPS, and particularly can inhibit the production of cytokines IL-6, IL-10, TNF-alpha and MCP-1 in serum of a test mouse in a sepsis model; and the survival rate of the test mice in the sepsis model is improved, and the body temperature and organ protection effects are exerted. In addition, the mejunin C in dihydro can inhibit the expression of dendritic cell surface co-stimulatory molecules (CD 80, CD86 and CD 40) and MHC class II molecules (Iab), and has the effects of inhibiting the activation of T cells, reducing cytokine storm and promoting the phagocytosis of bacteria by macrophages. Therefore, the mejunin C in dihydro can be used as an active ingredient for preparing anti-inflammatory and immunoregulatory medicines, and especially can be used for preparing anti-inflammatory and immunoregulatory medicines for treating sepsis, thereby relieving critical symptoms which are difficult to control in clinical treatment of sepsis, and having obvious clinical application value.
Claims (10)
1. An application of an active ingredient of an immunomodulator in preparing an anti-inflammatory medicament, which is characterized in that: the anti-inflammatory drug contains an immunomodulator active ingredient comprising one or more of mevalonate C in dihydro, stereoisomers of mevalonate C in dihydro, precursor compounds and pharmaceutically acceptable salts.
2. Use according to claim 1, characterized in that: the anti-inflammatory drug is used for treating sepsis.
3. Use according to claim 1, characterized in that: the anti-inflammatory drug improves the survival rate of sepsis and/or plays a role in protecting the body temperature and organs of sepsis.
4. Use according to claim 1, characterized in that: the anti-inflammatory agents also include antibiotics and/or hormones in combination with the immunomodulator active ingredients.
5. Use according to claim 4, characterized in that: the antibiotics are selected from one or more of carbapenem antibiotics and quinolone antibiotics.
6. Use according to claim 4, characterized in that: the antibiotic is meropenem or levofloxacin.
7. Use according to claim 4, characterized in that: the hormone is dexamethasone.
8. Use according to any one of claims 1-7, characterized in that: the anti-inflammatory medicament also comprises a pharmaceutically acceptable carrier and/or other auxiliary materials which do not influence the effectiveness of the active ingredients of the immunomodulator.
9. Use according to claim 8, characterized in that: the anti-inflammatory medicament is in the form of tablets, capsules, powder, granules, dripping pills, syrup, solution, suspension, injection, tincture, oral liquid, aerosol, buccal agent, medicinal granules, pills, powder or nano preparations.
10. Use of one or more of mevalonate C in dihydro, stereoisomers, precursor compounds, pharmaceutically acceptable salts of mevalonate C in dihydro for the preparation of an immunomodulator.
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2023
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