Application of piperidine acid in preparation of medicine for treating sepsis

Abstract

The invention provides application of piperidine acid in preparation of a medicine for treating sepsis, and relates to the technical field of new application of medicines. The invention finds that the piperidine acid can obviously improve the symptom of sepsis and has no obvious side effect. Therefore, the piperidine acid can be used for developing a novel medicine for treating sepsis, and has wide medical prospects and economic values.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of pipecolic acid in preparing a medicine for treating sepsis. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by an imbalanced host response to infection, with extremely high incidence and mortality rates. It is estimated that in 2017, 49 million patients worldwide suffered from sepsis events, and 11 million patients died from sepsis, accounting for 19.7% of all deaths worldwide. According to foreign epidemiological surveys, the mortality rate of sepsis has exceeded that of myocardial infarction, becoming the main cause of death for non-cardiac patients in intensive care units. Sepsis treatment is expensive, consumes a lot of medical resources, and seriously affects the quality of human life, posing a huge threat to human health. The pathogenesis of sepsis is extremely complex and has not yet been fully elucidated. So far, there is no effective targeted therapeutic drug. Sepsis treatment has become a major problem that needs to be solved urgently.

[0003] When the host suffers from sepsis, the host's body will be severely hit by damaging factors, resulting in damage to the body's immune cells, including macrophages, lymphocytes, and dendritic cells, and severe disorders in the immune functions of multiple organs and tissues. As a result, a large number of key immune effector cells in the host's important organs will die in the process. The death of immune effector cells will not only lead to a large number of immune cells, but also affect the normal function of immune cells. Therefore, sepsis-induced immune cell death will damage the host's immune defense capabilities. Therefore, sepsis may cause coagulation disorders, mitochondrial dysfunction, immune dysfunction, metabolic disorders, etc. in the host, causing the host to eventually suffer from multiple organ dysfunction syndrome or even death. However, with the prevalence of drug-resistant strains, existing clinical treatment methods have gradually fallen into trouble, and there is an urgent need to develop effective new drugs for the treatment of sepsis.

[0004] At present, there is no specific drug for the treatment of sepsis, and the drugs used in the treatment of sepsis are also very limited. The treatment of sepsis mainly relies on antibiotics to control infection, fluid resuscitation, and vasoactive drugs. Pipecolic acid is mainly used as a pharmaceutical intermediate and plant disease resistance activator, and there is no report on its efficacy in treating sepsis. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of pipecolic acid in the preparation of a medicament for treating sepsis.

[0006] To achieve the above object, the technical solution adopted by the present invention is: to provide the use of pipecolic acid in the preparation of a drug for treating sepsis.

[0007] Pipecolic acid generally refers to 2-piperidinic acid, with a molecular formula of C6H 11 NO2, currently, pipecolic acid is a non-protein amino acid widely distributed in plants, animals and microorganisms. It is an important precursor of many useful microbial secondary metabolites, such as rapamycin, antitumor agent swainsonine, peptide antibiotic virginiamycin and anthelmintic marcfortine. It is also a metabolite positively correlated with healthy diet and fruit and vegetable intake. It is also an intermediate mediator of the protective effect of long-term regular exercise on atherosclerosis. There is no report that pipecolic acid has the efficacy of treating sepsis.

[0008] The inventor unexpectedly discovered in the study that pipecolic acid can significantly improve the symptoms of sepsis without obvious side effects. Therefore, pipecolic acid can be used to develop new drugs for the treatment of sepsis, which has broad medical prospects and economic value.

[0009] As a preferred embodiment of the medicine provided by the present invention, the medicine includes a product for preparing to alleviate liver damage in patients with sepsis.

[0010] As a preferred embodiment of the drug provided by the present invention, the drug includes a product for preparing to reduce the expression level of inflammatory factors in macrophages of sepsis patients.

[0011] As a preferred embodiment of the medicine provided by the present invention, the pro-inflammatory factor includes at least one of TNF-α, IL-1β, and inducible nitric oxide synthase.

[0012] The present invention also provides a pharmaceutical composition for preventing and / or treating sepsis, the drug comprising pipecolic acid and a pharmaceutically acceptable excipient. The pharmaceutical composition for preventing and / or treating sepsis provided by the present invention applies pipecolic acid to sepsis, which not only provides a new source for preparing drugs for treating sepsis, but also discovers new medicinal value of pipecolic acid.

[0013] As a preferred embodiment of the pharmaceutical composition provided by the present invention, the effective concentration of pipecolic acid is 100 to 200 μg / kg.

[0014] As a preferred embodiment of the pharmaceutical composition provided by the present invention, the auxiliary materials include at least one of a filler, a disintegrant, a binder, a lubricant and a flavoring agent.

[0015] As a preferred embodiment of the pharmaceutical composition provided by the present invention, the dosage form of the drug includes at least one of injection, oral solution, pill, powder, paste, tablet, granule, powder or capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A is a schematic diagram of the experimental design for verifying that pipecolic acid can reduce sepsis-related inflammatory damage in mice at the animal level in Example 1 of the present invention; Figure 1 B is a graph showing the results of the concentration of pipecolic acid in mouse serum (n=8) after 1 hour of pipecolic acid pretreatment in Test Example 1 of the present invention; Figure 1 C is a graph showing the number of leukocytes in mouse serum 6 hours after LPS modeling in Test Example 1 of the present invention; Figure 1 D is a graph showing the number of granulocytes in mouse serum 6 hours after LPS modeling in Test Example 1 of the present invention; Figure 1 E is a graph showing the number of lymphocytes in mouse serum after 6 hours of LPS modeling in Test Example 1 of the present invention; Figure 1 F is a graph showing the number of monocytes in mouse serum 6 hours after LPS modeling in Test Example 1 of the present invention; Figure 1 G is a graph showing the sepsis severity score results of mice in Test Example 1 of the present invention at different time points (0, 2, 4, 6, 12, 24, 48 and 72 hours) of LPS modeling; Figure 1 H is a graph showing the test results of blood glucose levels at different time points (0, 2, 4, 6, 12, 24, 48 and 72 hours) of the test example 1 of the present invention during LPS modeling; Figure 1 I is a graph showing the weight change test results of Test Example 1 of the present invention at different time points (0, 2, 4, 6, 12, 24, 48 and 72 hours) of LPS modeling; Figure 1 J is a graph showing the test results of body temperature changes at different time points (0, 2, 4, 6, 12, 24, 48 and 72 hours) of the LPS modeling in Test Example 1 of the present invention; Figure 1 K is a graph showing the test result of serum TNF-α level (n=8) in Test Example 1 of the present invention 24 hours after LPS modeling; Figure 1 L is a graph showing the test results of serum IL-1β level (n=8) in Test Example 1 of the present invention 24 hours after LPS modeling; Figure 1 M is a graph showing the test result of serum IL-1Ra level (n=8) in Test Example 1 of the present invention 24 hours after LPS modeling; Figure 1 N shows the test result of eosin (H&E) staining of liver tissue in Test Example 1 of the present invention 48 hours after LPS modeling (scale bar is 20 μm); Figure 1 O shows the test results of Oil Red O staining of liver tissue in Test Example 1 of the present invention 48 hours after LPS modeling (scale bar is 20 μm) and statistical analysis results (n=6); Figure 1 P shows the test results of Oil Red O staining of liver tissue in Test Example 1 of the present invention 48 hours after LPS modeling (scale bar is 20 μm) and statistical analysis results (n=6); Figure 2 A is an observation diagram of the morphology of macrophages cultured in Test Example 2 of the present invention (scale bar is 20 μm); Figure 2 B is a graph showing the results of determining the number and percentage of macrophages induced in vitro by flow cytometry analysis in Test Example 2 of the present invention; Figure 2 C shows cells without the addition of F4 / 80 primary antibody as a negative control as determined by flow cytometry in Test Example 2 of the present invention; Figure 3 A shows the mRNA level of the pro-inflammatory cytokine TNF-α in BMDMs treated with different doses of pipecolic acid (5 μM, 10 μM, 20 μM) and 100 ng / mL LPS for 6 hours in Test Example 2 of the present invention (n=6); Figure 3 B shows the mRNA level of the pro-inflammatory cytokine IL-1β in BMDMs treated with different doses of pipecolic acid (5 μM, 10 μM, 20 μM) and 100 ng / mL LPS for 6 hours in Test Example 2 of the present invention (n=6); Figure 3 C shows the mRNA level of the pro-inflammatory cytokine iNOS in BMDMs treated with different doses of pipecolic acid (5 μM, 10 μM, 20 μM) and 100 ng / mL LPS for 6 hours in Test Example 2 of the present invention (n=6). DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the drawings in the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0039] Example 1

[0040] This example is used to verify that pipecolic acid can reduce sepsis-related inflammatory damage in mice at the animal level.

[0041] Among them, male C57BL / 6 mice were used as experimental subjects, and the sepsis model was simulated by intraperitoneal injection of lipopolysaccharides (LPS, a unique component in the cell wall of Gram-negative bacteria, which can cause sepsis after infecting the host). The data measured by injecting normal saline or pipecolic acid 1 hour before LPS injection were used as the normal saline control group and pipecolic acid control group, respectively, and the data measured by injecting normal saline or pipecolic acid 24 hours after LPS injection were used as the LPS model group and pipecolic acid treatment group, respectively.

[0042] Animal husbandry: Male C57BL / 6 mice (6 weeks old) were used for the experiment after a 1-2 week adaptation period. Six mice were placed in one cage, and the standard mouse feed and normal temperature drinking water that had been strictly sterilized were regularly replaced.

[0043] Animal modeling and drug administration: 1 hour in advance, mice were intraperitoneally injected with 200 μg / kg saline or pipecolic acid, followed by 2 mg / kg LPS. 24 hours later, mice were intraperitoneally injected with 200 μg / kg saline or pipecolic acid. Figure 1 As shown in A. The severity of sepsis (MSS, murine sepsis score) and pathological tissue damage in mice were evaluated at 0 hours, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours and 72 hours after LPS modeling, and the pathological changes in body weight, blood sugar and body temperature, the early response of leukocytes, granulocytes, lymphocytes and monocytes, and the levels of serum pro-inflammatory cytokines and anti-inflammatory cytokines were detected.

[0044] Test Example 1

[0045] 1. Test Method

[0046] (1) Establishment of mouse sepsis model and assessment of disease severity

[0047] The sepsis model mice were established by a single intraperitoneal injection of ultrapure Escherichia coli O55:B5 strain LPS (2 mg / kg bodyweight). To monitor the disease process, the murine sepsis score (MSS: mainly including spontaneous activity, response to touch and auditory stimulation, posture, respiratory rate and quality, and appearance-the degree of hair erection), body weight, blood glucose, body temperature and other indicators were monitored at 8 different time points after LPS injection: 0h (equivalent to the baseline state), 2h, 4h, 6h, 12h, 24h, 48h and 72h. The mice were killed at 0h, 24h, 48h and 72h after LPS treatment, and the blood was collected in 1.5mL centrifuge tubes. After clotting at room temperature for 30min, the coagulated blood was centrifuged at 1500g for 10min to obtain serum for cytokine detection. At the same time, the liver tissue of the mice was isolated and collected, fixed with 4% paraformaldehyde solution, or quickly frozen in liquid nitrogen for biochemical analysis.

[0048] (2) Determination of pipecolic acid concentration

[0049] The concentration of pipecolic acid was determined by LC-MS / MS targeting of API 4000. The detection data were calculated and analyzed by ChemView software (Applied Biosystems, Inc., USA). The external standard curve correction method was used to calculate the concentration of metabolites in the sample based on the abundance ratio of metabolites to the corresponding external standards and the internal quality control product correction.

[0050] (3) Whole blood cell analysis

[0051] 20 μL of blood samples were collected from the mouse tail vein using a disposable micro-anticoagulant tube containing EDTA and analyzed using a five-differential automatic blood analyzer within 6 hours of blood collection.

[0052] (4) Specific cytokine detection

[0053] Specific cytokines TNF-α, IL-1β, and IL-1RA levels were detected using specific ELISA kits.

[0054] 1) Take out the previously packaged serum from the -80℃ freezer (50μL per sample, diluted 1:2 for testing) and thaw it on ice.

[0055] 2) After the various reagents are moved to room temperature and equilibrated for 30 minutes, prepare the reagents to be used:

[0056] ① Take out a standard sample, centrifuge it at 5000rpm at room temperature for 30s, and then dissolve it with 1mL of sample diluent. During this process, the pipette tip should be pointed at the bottom of the centrifuge tube and gently blow it repeatedly until it dissolves, finally obtaining the standard sample S7.

[0057] ②Take 7 1.5mL centrifuge tubes, mark them as S0-S6, arrange them in reverse order, and add 250μL of sample diluent to each centrifuge tube. First, pipette 250μL of standard S7 into the first centrifuge tube (S6), and mix it carefully by blowing. Next, pipette 250μL from S6 into the second centrifuge tube (S5), and mix it carefully by blowing. This method is used to perform multiple dilutions of the standard, with S0 as the sample diluent. The standard concentrations of TNF-α, IL-1β, and IL-1Ra are shown in Table 1, Table 2, and Table 3, respectively.

[0058] Table 1. TNF-α standard concentration

[0059]

[0060] Table 3. IL-1Ra standard concentrations

[0061]

[0062] 3) Sample addition: You need to set up sample wells and standard wells. Add 100 μL of diluted sample or standard to each well, shake gently to mix, cover the plate and incubate at 37°C for 2 hours (5 minutes before the end, dilute the biotin-labeled antibody at 1:100).

[0063] 4) Pour out the liquid in the wells and shake them dry with a little force. No washing is required in this step.

[0064] 5) Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour (5 minutes before the end, dilute the washing working solution at 1:25).

[0065] 6) Pour out the liquid in the wells, shake it dry, and then turn it upside down on a clean paper towel and pat it 2-3 times to ensure that there is no liquid residue in the wells (otherwise it will affect subsequent results). Wash the plate 3 times in this way, soaking it for 2 minutes each time, generally 300μL / well (fill it up, otherwise liquid will easily remain on the well wall), and shake it dry (during the washing process, take the time to dilute the horseradish peroxidase-labeled avidin at 1:100).

[0066] 7) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, attach a new plate sticker, and incubate at 37°C for 1 h.

[0067] 8) Pour out the liquid in the wells, shake it dry, then turn it upside down on a clean paper towel and pat it 2-3 times to ensure that there is no liquid residue in the wells (otherwise it will affect subsequent results). Wash the plate 5 times in this way, soaking it for 2 minutes each time, generally 300μL / well (fill it up, otherwise liquid will easily remain on the well wall), and shake it dry.

[0068] 9) Add 90 μL of substrate solution to each sample well in the order of adding samples previously, and develop color at 37°C in the dark for about 15 minutes (the color of the reaction solution needs to be checked during the process).

[0069] 10) Add 50 μL of stop solution to each sample well in order to terminate the color development reaction.

[0070] 11) After the reaction is terminated, preferably within 5 minutes, use an ELISA reader to measure the optical density of each well at a wavelength of 450 nm.

[0071] 12) A standard curve was prepared based on the optical density value of the standard and the corresponding concentration, and the standard curve formula was obtained to further calculate the concentration (pg / mL) of each sample.

[0072] (5) Histopathological analysis

[0073] Fresh liver and lung tissues were quickly frozen or fixed with 4% paraformaldehyde for more than 24 hours. The tissues were then embedded and sliced ​​to a thickness of 5 μm. The slices were then dewaxed using conventional methods. Hematoxylin and eosin (HE) staining was further performed according to the instructions of the kit. In addition, frozen liver sections prepared using optimal cutting temperature (OCT) compounds were stained with oil red and lipid droplet accumulation in the liver was observed. Histopathological staining images were taken using a microscope.

[0074] H&E staining: The experiment was performed according to the steps 1) to 4) below.

[0075] 1) Preparation of tissue pathology sections: After the tissue is fixed with 4% paraformaldehyde overnight, it is placed in a dehydrator with gradient concentrations of alcohol for dehydration, transparent in xylene, and fully immersed in liquid paraffin. Then, the specimen is clamped and placed in a wax mold with the cut surface facing down, and immediately embedded in wax after it is arranged neatly. Use a paraffin slicer to cut the embedded tissue block into thin slices of about 5μm thick, immediately put it in 60℃ warm water to flatten it, and then stick it on a slide, and then dry it for use.

[0076] 2) Dewaxing and hydration: The sections were sequentially treated with xylene I and xylene II for 30 min each, and with gradient alcohols of decreasing concentration for 10 min each (absolute ethanol 3 times, 95% ethanol 2 times, 90% ethanol, 80% ethanol, 70% ethanol), and finally washed with tap water for 5 min.

[0077] 3) Hematoxylin staining of nuclei and eosin staining of cytoplasm: Place tissue sections in hematoxylin staining for 5-10 minutes, then wash with tap water. Next, immerse the sections in a dilute hydrochloric acid alcohol solution for color separation for a few seconds, then wash with tap water. Immerse the sections in dilute ammonia water to blue the nuclei for 3-5 minutes, then wash with tap water again. Finally, immerse in eosin staining solution for 5-10 minutes, then wash with tap water.

[0078] 4) Dehydration, transparency and sealing: Then, the washed tissue sections are dehydrated in gradient alcohol for 1 min each (according to the concentration from low to high, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol twice, and anhydrous ethanol three times). Transparentize after 5 min each in xylene I and xylene II. Add neutral gum to the slide for sealing, and be careful not to generate bubbles. After sealing, dry in a fume hood and examine under a microscope.

[0079] Oil Red O staining: Perform the experiment according to the steps 1) to 4) below.

[0080] 1) Preparation of working solution: Mix Oil Red O stock solution and deionized water in a ratio of 6:4, let stand at room temperature for 10 minutes, and then filter it with a 0.22μm filter membrane. Note: The freshly prepared working solution must be used within 2 hours;

[0081] 2) After the liver tissue is dehydrated, it is embedded in a tissue embedding agent and then frozen and sectioned to a thickness of about 10 μm. The sections are then attached to an adhesive slide and left to dry overnight at room temperature. The next day, the sections are rinsed with running tap water for 5 minutes and then stained with Oil Red O.

[0082] 3) After rinsing the sections with 60% isopropanol for 15 seconds, stain the sections with freshly prepared Oil Red O working solution for 15 minutes, and then rinse the sections with 60% isopropanol for 30 seconds;

[0083] 4) Dip the slides in hematoxylin stain solution for 5 times to slightly stain the cell nuclei, rinse with distilled water, dry at room temperature, and then seal the slides with a water-soluble sealing medium. Observe the staining results under an ordinary optical microscope.

[0084] Immunofluorescence staining: Perform the experiment according to the steps 1) to 5) below.

[0085] 1) Preparation of tissue pathology sections: After the tissue is fixed with 4% paraformaldehyde overnight, it is placed in a dehydrator with gradient concentrations of alcohol for dehydration, transparent in xylene, and fully immersed in liquid paraffin. Then, the specimen is clamped and placed in a wax mold with the cut surface facing down, and immediately embedded in wax after it is arranged neatly. Use a paraffin slicer to cut the embedded tissue block into thin slices of about 5μm thick, immediately put it in 60℃ warm water to flatten it, and then stick it on a slide, and then dry it for use.

[0086] 2) Dewaxing and hydration: The sections were sequentially treated with xylene I and xylene II for 30 min each, and with gradient alcohols of decreasing concentration for 10 min each (absolute ethanol 3 times, 95% ethanol 2 times, 90% ethanol, 80% ethanol, 70% ethanol), and finally washed with tap water for 5 min.

[0087] 3) Antigen repair: Place the slides on a metal staining rack and perform antigen repair on the sections in a 95°C water bath using 1× sodium citrate buffer for 20 minutes. After the sections cool naturally, rinse with PBS for 10 minutes.

[0088] 4) Blocking and immune response 1: Use a wax pen to draw circles close to the tissue, add an appropriate amount of 0.1% Triton-X 100 to the specimen, incubate at room temperature for 15 minutes, gently remove the Triton, then add normal goat serum as a blocking solution, and block at room temperature for 1 hour. Shake off the blocking solution with a little force, use filter paper to absorb the excess liquid around the specimen, add rabbit anti-mouse F4 / 80 antibody (1:400) to each slice of tissue, and place it in a wet box in a refrigerator at 4°C overnight. The next day, take out the wet box and slices, shake off the primary antibody, wash with PBS 3 times, each time for 5 minutes, then use filter paper to carefully absorb the liquid around the tissue, and finally add Alexa 555-labeled goat anti-rabbit IgG (1:300) was incubated at room temperature for 1 h.

[0089] 5) Sealing: shake off the secondary antibody, wash the tissue sections with PBST for 5 min × 3 times, stain the nucleus with DAPI for 5 min, wash the tissue sections again with PBST for 5 min × 3 times, add anti-fade mounting agent to seal the sections, let them dry at 4°C in the dark, and observe and collect images with a confocal microscope.

[0090] 2. Test results and analysis

[0091] First, compared with the normal saline control group mice, the serum concentration of pipecolic acid in the pipecolic acid-treated group was significantly increased (p < 0.001; e.g. Figure 1 At the same time, in the early stage of LPS inflammation - 6 hours after LPS modeling, compared with the normal saline control group mice, the white blood cells in the blood of the mice treated with pipecolic acid (such as Figure 1 C), granulocytes (as shown in Figure 1 D), lymphocytes (as shown in Figure 1 E) and monocytes (as Figure 1 Compared with the normal saline control group, the MSS ( Figure 1 G), pathological hypoglycemia (such as Figure 1H), weight loss (as shown in Figure 1 I) and hypothermia (as Figure 1 J) recovery was accelerated; at the same time, 24 hours after LPS modeling, compared with the normal saline control group mice, the serum TNF-α (as shown in Figure 1 K) levels and IL-1β levels (as Figure 1 L) was significantly decreased, while IL-1Ra (as shown in Figure 1 M) levels were significantly increased, and the differences were statistically significant (p<0.05). In addition, compared with the mice in the normal saline control group, inflammatory cell infiltration and lipid droplet aggregation in the liver of mice in the pipecolic acid treatment group were significantly reduced 48 hours after LPS modeling (as shown in Figure 2). Figure 1 N. Figure 1 P). In this test example, the values ​​are expressed as mean ± SEM (non-statistically significant is expressed as ns, p>0.05, *p<0.05, **p<0.01, ***p<0.001).

[0092] The above results indicate that, at the animal level, pretreatment with pipecolic acid can protect multiple organ tissue pathological damage in LPS-induced sepsis, reduce the severity of sepsis and promote its recovery. Therefore, the above observations confirm that pipecolic acid intervention in vivo can reduce sepsis-related inflammatory damage in mice.

[0093] Example 2

[0094] This example is used to explore the effect of pipecolic acid on the pro-inflammatory response of macrophages in septic mice.

[0095] Among them, macrophage colony stimulating factor (M-CSF, 20ng / mL) was used to induce bone marrow progenitor cells to differentiate into macrophages (marrow-derived macro phages, BMDMs) as the experimental subjects. Phosphate buffered saline (PBS) was used as the control group; BMDMs cells were added with 100ng / mL of LPS, and the LPS treatment group was not added with pipecolic acid, and the pipecolic acid treatment group was added with different doses of pipecolic acid (5μM, 10μM, 20μM). The mRNA expression levels of inflammatory factors (mainly including TNF-α, IL-1β and inducible nitric oxide synthase iNOS) in the above-mentioned treatment groups and the control group were detected.

[0096] Test Example 2

[0097] 1. Test Method

[0098] (1) Isolation and differentiation of mouse bone marrow-derived macrophages

[0099] 1) 6-10 week old mice were killed by dislocation and disinfected in 75% ethanol for 5 min.

[0100] 2) Use autoclaved instruments in a clean bench to carefully remove the femur and tibia of the mouse (do not cut them at this time), carefully separate the muscles and cut the two ends of the bones to expose the bone marrow cavity, use a 10mL sterile syringe to absorb PBS and then change the needle of a 1mL syringe to gently and slowly flush the bone marrow cavity cells into a 50mL centrifuge tube, and rinse repeatedly until the bone turns white (generally 10mL of flushing solution is required for each bone).

[0101] 3) Use a 70 μm filter to remove tissue clumps and centrifuge at 1000 rpm for 5 min.

[0102] 4) Discard the supernatant, aspirate 3 mL of red blood cell lysis buffer to resuspend the cells, let stand at room temperature for 5 minutes, then add 3 volumes of PBS to stop red blood cell lysis, and centrifuge at 1000 rpm for 5 minutes.

[0103] 5) Discard the supernatant, resuspend the bone marrow cells in DMEM medium containing 20 ng / mL M-CSF, 10% FBS and 1% penicillin / streptomycin, and evenly inoculate them into 10 cm culture dishes (based on experience, bone marrow cells from one mouse can be inoculated in 5-6 10 cm culture dishes). Supplement each dish with 10 mL of medium containing M-CSF to stimulate the differentiation of bone marrow cells into monocytes and macrophages.

[0104] 6) After culturing for 24 h, add 10 mL of fresh medium containing M-CSF to each dish. On the 5th day, half of the medium (containing 20 ng / mL M-CSF) was replaced. On the 7th day, the induction was terminated. The induced macrophages were digested with trypsin, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then, the cells were resuspended in DMEM containing 10% FBS and plated at 2×10 6 / mL was inoculated in a 6-well plate, 2mL per well, and corresponding treatment was carried out the next day.

[0105] (2) Identification of BMDMs

[0106] At the same time, the induced differentiated macrophages on the 7th day were identified according to the morphology under light microscope and characteristic surface molecular markers. The induced macrophages were digested with trypsin, centrifuged and resuspended with 100 μL 5% FBS-PBS flow cytometry fluid to the required number of cells after cell counting, and APC anti-mouse F4 / 80 (1:100 dilution) fluorescent antibody was added. The cells were incubated on ice in the dark for 30 min, centrifuged at 4°C, 1500 rpm for 5 min, washed once with 1 mL 5% FBS-PBS flow cytometry fluid, resuspended with 200 μL 5% FBS-PBS flow cytometry fluid, and detected by the machine. The F4 / 80 positive cell population was observed and counted.

[0107] 2. Test results and analysis

[0108] The morphology of mouse bone marrow-derived macrophages (BMDMs) under a white light microscope is as follows Figure 2 As shown in A, where Figure 2 As shown in A, the BMDMs induced in this example are rhombic or irregular in shape and can extend pseudopodia.

[0109] Mouse EGF-like module-containingmucin-like hormone receptor-like 1 (EMR1, also known as F4 / 80) is a cell surface glycoprotein and a member of the EGFTM7 protein family. It has 68% amino acid identity with the human EMR1 protein. Studies have found that during the maturation and activation of macrophages, the expression of F4 / 80 protein changes significantly. Therefore, by observing the expression of F4 / 80 in M-CSF-induced BMDMs, the purity of in vitro induced macrophages can be measured. The identification results obtained by flow cytometry showed that the purity of the macrophages induced in vitro in Example 2 reached 97.33% (such as Figure 2 B. As shown in 2C).

[0110] Furthermore, at the cellular level, BMDMs were treated with different doses of pipecolic acid (5μM, 10μM, 20μM) and LPS (100ng / mL) for 6 hours, and then the cells were collected and the mRNA levels of inflammatory factors (mainly including TNF-α, IL-1β and inducible nitric oxide synthase iNOS) were detected. The results showed that pipecolic acid inhibited the levels of proinflammatory cytokines TNF-α, IL-1β and iNOS induced by LPS in a concentration-dependent manner (p<0.05; as shown in Figure 2). Figure 3 A. Figure 3 B. Figure 3 C, where *p<0.05, ***p<0.001).

[0111] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of pipecolic acid in the preparation of a medicament for treating sepsis.

2. The drug according to claim 1, characterized in that The drugs include drugs for alleviating liver damage in patients with sepsis.

3. The drug according to claim 1, characterized in that The drugs include drugs for reducing the expression level of pro-inflammatory cytokines in macrophages of patients with sepsis.

4. The drug according to claim 3, characterized in that The pro-inflammatory cytokines include at least one of TNF-α, IL-1β, and inducible nitric oxide synthase.

5. A pharmaceutical composition for preventing and / or treating sepsis, characterized in that: The pharmaceutical composition comprises the drug according to claims 1 to 4 and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that The concentration of the pipecolic acid is 100-200 μg / kg.

7. The pharmaceutical composition according to claim 5, characterized in that The auxiliary materials include at least one of a filler, a disintegrant, a binder, a lubricant and a flavoring agent.

8. The pharmaceutical composition according to claim 5, characterized in that The dosage form of the drug includes at least one of injection, oral solution, pill, powder, paste, tablet, granule, powder or capsule.

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